Figure 2Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264 . 7 cells after LPS stimulation ( A ) . Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation . The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount ( B ) . Effect of Okadaic acid ( OA ; 100nM ) , the PP2A inhibitor , on miRNA activity . Change in fold repression for a let - 7a reporter upon LPS exposure in control and OA treated cells ( mean + / - s . e . m . , n = 3 ) ( C ) . Schematic representation of the in vitro phosphatase assay ( upper panel ) . PP2A was immunoprecipitated from 24hrs LPS treated RAW 264 . 7 cells and were incubated in vitro with wild type or phosphorylation defective FH - Ago2 mutant ( Ago2Y529F ) isolated from HEK293 cells transfected with respective expression constructs . Phosphorylated Ago2 ( pYAgo2 ) levels were measured by western blot analysis using anti - phospho Tyrosine specific 4G10 antibody . Relative intensities were quantified by densitometric analysis and mentioned below the respective panels . FH - Ago2 was detected by anti - HA antibody ( Lower panel ) . Defective de novo miRNP formation in cells pre - treated with PP2A inhibitor . Ago2 associated ( F ) and total ( G ) miR - 122 levels were measured by qRT - PCR and normalized against immuneprecipitated FH - Ago2 content and U6 RNA respectively . Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted . All samples were treated for 24 hrs with LPS ( mean + / - s . e . m . , n = 4 ) . PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Expression of PTPA after LPS stimulation in RAW 264 . 7 cells as well as in PEC isolated from BALB / c mice . β - actin is used as loading control ( H ) . PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2 . Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264 . 7 cells ( I ) . Phosphorylated Ago2 level was measured by western blot in HA - immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH - Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody ( I , left panel ) . The PTPA was detected with anti PTPA antibody , β - Actin was used as loading control ( I , right panel ) . Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs . Ago2 - associated miRNA ( J , left panel ) ( mean + / - s . e . m . , n = 3 ) and mRNA levels ( J , right panel ) ( mean + / - s . e . m . , n = 2 ) were also quantified using qRT - PCR . PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264 . 7 cells treated with LPS for 24 hrs and was incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells and upon the assay reaction , the phospho - Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody ( K , right panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor Expression of PP2A was detected in western blot done for cell extract from control and OA treated RAW 264.7 cells after LPS stimulation (A).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on Ago2 phosphorylation The amount of phosphorylated Ago2 was measured in OA treated cells before and after LPS stimulation. The amount of Tyr phosphorylated Ago2 was measured by densitometric quantification of western blot data upon normalization against immunoprecipitated Ago2 amount (B).Effect of Okadaic acid (OA; 100nM), the PP2A inhibitor, on miRNA activity. Change in fold repression for a let-7a reporter upon LPS exposure in control and OA treated cells (mean+/- s.e.m., n=3) (C).Schematic representation of the in vitro phosphatase assay (upper panel). PP2A was immunoprecipitated from 24hrs LPS treated RAW 264.7 cells and were incubated in vitro with wild type or phosphorylation defective FH-Ago2 mutant (Ago2Y529F) isolated from HEK293 cells transfected with respective expression constructs. Phosphorylated Ago2 (pYAgo2) levels were measured by western blot analysis using anti-phospho Tyrosine specific 4G10 antibody. Relative intensities were quantified by densitometric analysis and mentioned below the respective panels. FH-Ago2 was detected by anti-HA antibody (Lower panel).Defective de novo miRNP formation in cells pre-treated with PP2A inhibitor. Ago2 associated (F) and total (G) miR-122 levels were measured by qRT-PCR and normalized against immuneprecipitated FH-Ago2 content and U6 RNA respectively. Values obtained upon 14 hrs of DOX treatment were considered as unit and relative values obtained upon 24 hrs of DOX treatment are plotted. All samples were treated for 24 hrs with LPS (mean+/- s.e.m., n=4).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Expression of PTPA after LPS stimulation in RAW 264.7 cells as well as in PEC isolated from BALB/c mice. β-actin is used as loading control (H).PTPA is an essential factor for PP2A mediated dephosphorylation of Ago2. Effect of PTPA depletion on Ago2 phosphorylation level in RAW 264.7 cells (I). Phosphorylated Ago2 level was measured by western blot in HA-immunoprecipitated materials isolated from either siCon or siPTPA transfected cells expressing FH-Ago2 upon 24 hrs of LPS treatment using a phosphotyrosine specific 4G10 antibody (I, left panel). The PTPA was detected with anti PTPA antibody, β-Actin was used as loading control (I, right panel).Effect of PTPA depletion on Ago2 associated miRNAs and mRNAs. Ago2-associated miRNA (J, left panel) (mean+/- s.e.m., n=3) and mRNA levels (J, right panel) (mean+/- s.e.m., n=2) were also quantified using qRT-PCR.PP2A was immunoprecipitated from siCon or siPTPA treated RAW 264.7 cells treated with LPS for 24 hrs and was incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells and upon the assay reaction, the phospho-Ago2 level was measured by western blot analysis using phosphotyrosine specific 4G10 antibody (K, right panel).


Figure 3Expression of different cytokines and phosphatases in RAW 264 . 7 cells and PEC infected with Leishmania donovani ( Ld ) parasites . Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264 . 7 ( B , left panel ) and mouse peritoneal macrophage cells PEC ( B , right panel ) . Cytokine mRNA levels like IL - 10 , TNF - α and IL - 6 were quantified after 24 hrs of Ld infection in RAW 264 . 7 ( C , upper panel mean + / - s . e . m . , n = 3 ) and mouse peritoneal macrophage PEC ( mean + / - s . e . m . , n = 3 ) ( C , Lower panel ) . Effect of Ld infection on Ago2 phosphorylation and its miRNA association . RAW 264 . 7 cells were transfected with FH - Ago2 expression construct and infection was done for various time points followed by FH - Ago2 pull down using anti - FLAG beads . Phosphorylated Ago2 levels were checked during the course of Ld infection ( D ) . Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let - 7a level measurement in primary macrophages ( mean + / - s . e . m . , n = 2 ) ( E , left panel ) and in RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) ( E , right panel ) . Schematic representation of in vitro phosphatase assay ( F , upper panel ) . PP2A , MKP1 , MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody ( F , lower panel ) and measured by densitometry . BC ; Bead Control . PP2A MKP3 , or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH - Ago2 isolated from FH - Ago2 stable HEK293 cells . Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted ( mean + / - s . e . m . , n = 3 ) ( G ) . BC ; Bead Control . Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells ( H ) . Effect of PP2A knockdown on Ago2 phosphorylation and miRNA - Ago2 binding in RAW 264 . 7 cells infected with Ld . Cells were co - transfected with siRNA and FH - Ago2 expression plasmid . Ago2 associated let - 7a levels were also estimated and plotted ( mean + / - s . e . m . , n = 4 ) ( I ) . Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment ( 100 nM ) were detected . Cells were pre - treated with OA ( 100 nM ) for 2 hrs before infection ( J ) . β - Actin was used as loading control . Effect of OA on Ago2 phosphorylation in Ld - infected RAW 264 . 7 cells and mouse PEC . Phospho - Ago2 level was measured in OA pre - treated ; Ld infected RAW 264 . 7 cells expressing FH - Ago2 and in PEC post 24 hrs of infection with or without OA pre - treatment . Phosphorylated Ago2 levels were also checked ( K ) . Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots . Alix was used at MVB marker , while Calnexin was used as ER marker ( L ) . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . OptiprepR fractions , positive for MVB ( fraction number 2 , 3 , 4 ) and ER ( fraction number 7 , 8 , 9 ) markers were pooled separately and Ago2 was immunoprecipitated using Anti - FLAG beads . Phosphorylated Ago2 levels were quantified in western blot done with phospho - tyrosine specific 4G10 antibody ( M ) . Subcellular compartmentalization of PP2A in Ld - infected RAW 264 . 7 cells . RAW 264 . 7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures . PP2A percentage intensity plot for each fraction was done for control and infected samples ( N ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Expression of different cytokines and phosphatases in RAW 264.7 cells and PEC infected with Leishmania donovani (Ld) parasites. Cells at different time points of infection were harvested and levels of various phosphatases were checked by western blot analysis using antibodies specific for different phosphatases in both RAW 264.7 ( B, left panel) and mouse peritoneal macrophage cells PEC (B, right panel).Cytokine mRNA levels like IL-10, TNF-α and IL-6 were quantified after 24 hrs of Ld infection in RAW 264.7 (C, upper panel mean +/- s.e.m., n=3) and mouse peritoneal macrophage PEC (mean+/- s.e.m., n=3) (C, Lower panel).Effect of Ld infection on Ago2 phosphorylation and its miRNA association. RAW 264.7 cells were transfected with FH-Ago2 expression construct and infection was done for various time points followed by FH-Ago2 pull down using anti-FLAG beads. Phosphorylated Ago2 levels were checked during the course of Ld infection (D).Phosphorylation levels of Ago2 were measured by doing endogenous Ago2 pull down using Ago2 specific antibody after 4 hrs of infection and quantified along with Ago2 associated let-7a level measurement in primary macrophages (mean+/- s.e.m., n=2) (E, left panel) and in RAW 264.7 cells (mean+/- s.e.m., n=3) (E, right panel) .Schematic representation of in vitro phosphatase assay (F, upper panel). PP2A, MKP1, MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Phosphorylated Ago2 level was detected by western blot analysis using phosphotyrosine specific 4G10 antibody (F, lower panel) and measured by densitometry. BC; Bead Control.PP2A MKP3, or SHP1 were immunoprecipitated individually from naïve or Leishmania infected macrophage cells and were incubated in vitro with FH-Ago2 isolated from FH-Ago2 stable HEK293 cells. Relative intensity of 4G10 specific band against immunoprecipitated Ago2 was plotted (mean+/- s.e.m., n=3) (G). BC; Bead Control.Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Phosphorylated Ago2 levels were detected after 4 hrs of infection in siCon or siPP2A transfected cells (H).Effect of PP2A knockdown on Ago2 phosphorylation and miRNA-Ago2 binding in RAW 264.7 cells infected with Ld. Cells were co-transfected with siRNA and FH-Ago2 expression plasmid. Ago2 associated let-7a levels were also estimated and plotted (mean+/- s.e.m., n=4) (I).Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells Cellular levels of PP2A and Ago2 at different time points of Ld infection with or without OA treatment (100 nM) were detected. Cells were pre-treated with OA (100 nM) for 2 hrs before infection (J). β-Actin was used as loading control.Effect of OA on Ago2 phosphorylation in Ld-infected RAW 264.7 cells and mouse PEC. Phospho-Ago2 level was measured in OA pre-treated; Ld infected RAW 264.7 cells expressing FH-Ago2 and in PEC post 24 hrs of infection with or without OA pre-treatment. Phosphorylated Ago2 levels were also checked (K).Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. Subcellular localization of Ago2 and PP2A in individual fractions were detected by western blots. Alix was used at MVB marker, while Calnexin was used as ER marker (L).RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. OptiprepR fractions, positive for MVB (fraction number 2,3,4) and ER (fraction number 7,8,9) markers were pooled separately and Ago2 was immunoprecipitated using Anti-FLAG beads. Phosphorylated Ago2 levels were quantified in western blot done with phospho-tyrosine specific 4G10 antibody (M).Subcellular compartmentalization of PP2A in Ld-infected RAW 264.7 cells. RAW 264.7 cells were infected with Ld for 6 hrs and cell extract were analysed on a OptiprepR density gradient to separate subcellular organelles and structures. PP2A percentage intensity plot for each fraction was done for control and infected samples (N).


Figure 4Effect of PP2A downregulation or inhibition on pro and anti - inflammatory cytokine levels in Ld infected macrophage . IL - 10 levels were measured after Ld infection in PP2A knocked - down RAW 264 . 7 cells ( mean + / - s . e . m . , n = 3 ) and in PEC ( mean + / - s . e . m . , n = 3 ) . IL - 6 was also measured in RAW cells after OA treatment ( mean + / - s . e . m . , n = 5 ) and also in PEC pre - treated with OA ( mean + / - s . e . m . , n = 3 ) . Values obtained at 0 hrs ( non - infected ) or non - OA treated samples were considered as unit in each case . IL - 6 protein level ( n = 5 ) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock - down RAW 264 . 7 cells . Effect of PP2A knock - down on parasite internalization Internalized parasites were imaged in RAW 264 . 7 cells stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 ( red ) ( B ) . Effect of PP2A knock - down on parasite internalization and cellular IL - 1β level . Number of Ld internalized was measured in control and PP2A knock - down cells and relative numbers of parasites within infected cells were plotted ( C , left panel , ( mean + / - s . e . m . , n = 20 cells ) . IL - 1β mRNA levels were also quantified in those cells by qRT - PCR done for total cellular RNA [ C , right panel , ( mean + / - s . e . m . , n = 3 ) ] . Scale bar 20 µm . 4x Zoomed insets are shown . Effect of PP2A knockdown on IL - 10 level in Ld - infected cells expressing phosphorylation defective mutant of Ago2 . In RAW 264 . 7 cells , depleted for endogeneous Ago2 ( using a specific siRNA against the 3 ' UTR of Ago2 mRNA ) , either the wild type or Y529F mutant of FH - Ago2 ( without having the 3 ' UTR of Ago2 ) was expressed in siCon or siPP2A treated RAW 264 . 7 cells . IL - 10 mRNA level was measured subsequent to Ld infection . Relative IL - 10 levels were plotted . Expression levels in non - infected siCon treated cells in each case were designated as unit ( mean + / - s . e . m . , n = 3 ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . In images obtained , Ago2 was detected with α - HA ( detected at 405nm ) and Leishmania was stained for GP63 ( detected at 564nm ) ( E ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . In RAW 264 . 7 cells transfected with FH - Ago2 WT and Y529F mutant and pre - treated with OA , Leishmania internalization was measured microscopically . Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted . Scale bar 10 µm . RNA was isolated from different experimental sets and TNF - α , IL - 1β and IL - 10 mRNA levels were estimated ( mean + / - s . e . m . , n = 3 ) ( F ) . Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264 . 7 cells . RAW 264 . 7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs . RAW 264 . 7 cells were stained with Phalloidin - Alexa 488 ( green ) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 ( red ) ( G , left panel ) . Number of internalized parasites per 100 macrophages were calculated and relative values were plotted ( G , right panel ) ( mean + / - s . e . m . , n = 20 ) . Scale bar 20 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Effect of PP2A downregulation or inhibition on pro and anti-inflammatory cytokine levels in Ld infected macrophage. IL-10 levels were measured after Ld infection in PP2A knocked-down RAW 264.7 cells (mean+/- s.e.m., n=3) and in PEC (mean+/- s.e.m., n=3). IL-6 was also measured in RAW cells after OA treatment (mean+/- s.e.m., n=5) and also in PEC pre-treated with OA (mean+/- s.e.m., n=3). Values obtained at 0 hrs (non-infected) or non-OA treated samples were considered as unit in each case. IL-6 protein level (n=5) in cell supernatant was also measured by ELISA after Ld infection in PP2A knock-down RAW 264.7 cells.Effect of PP2A knock-down on parasite internalization Internalized parasites were imaged in RAW 264.7 cells stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton detection and Ld was detected by indirect immunofluorescence done for parasite specific membrane protein GP63 (red) (B).Effect of PP2A knock-down on parasite internalization and cellular IL-1β level. Number of Ld internalized was measured in control and PP2A knock-down cells and relative numbers of parasites within infected cells were plotted (C, left panel, (mean+/- s.e.m., n=20 cells). IL-1β mRNA levels were also quantified in those cells by qRT-PCR done for total cellular RNA [C, right panel, (mean+/- s.e.m., n=3)]. Scale bar 20 µm. 4x Zoomed insets are shown.Effect of PP2A knockdown on IL-10 level in Ld-infected cells expressing phosphorylation defective mutant of Ago2. In RAW 264.7 cells, depleted for endogeneous Ago2 (using a specific siRNA against the 3'UTR of Ago2 mRNA), either the wild type or Y529F mutant of FH-Ago2 (without having the 3'UTR of Ago2) was expressed in siCon or siPP2A treated RAW 264.7 cells. IL-10 mRNA level was measured subsequent to Ld infection. Relative IL-10 levels were plotted. Expression levels in non-infected siCon treated cells in each case were designated as unit (mean+/- s.e.m., n=3).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. In images obtained, Ago2 was detected with α-HA (detected at 405nm) and Leishmania was stained for GP63 (detected at 564nm) (E).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. In RAW 264.7 cells transfected with FH-Ago2 WT and Y529F mutant and pre-treated with OA, Leishmania internalization was measured microscopically. Quantitative measurement of GP63 positive structures was done and quantitative data per 100 infected cells was plotted. Scale bar 10 µm. RNA was isolated from different experimental sets and TNF-α, IL-1β and IL-10 mRNA levels were estimated (mean +/- s.e.m., n=3) (F).Effect of PP2A inhibitor OA on Ld internalization and cytokine production in RAW 264.7 cells. RAW 264.7 cells with and without OA treatment were given Ld infection for 2 hrs and 24 hrs. RAW 264.7 cells were stained with Phalloidin-Alexa 488 (green) for actin cytoskeleton and Ld was stained for parasite specific protein GP63 (red) (G, left panel). Number of internalized parasites per 100 macrophages were calculated and relative values were plotted (G, right panel) (mean+/- s.e.m., n=20). Scale bar 20 µm.


Figure 5LPS induced miRNA export from mammalian macrophage cells . A schematic diagram of the experiments has been described in left panel of ( A ) . Characterization and quantification of exosomes derived from Control and LPS - activated RAW 264 . 7 cells by Nanoparticle Tracking Analysis ( NTA ) done for isolated exosomes . Relative size distributions are shown ( A , right panel ) . Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264 . 7 cells . RNA content was normalized against total exosomal proteins ( B ) . β - Actin as loading control for HuR western blot . Binding of miRNAs with HuR after LPS exposure was measured . Amount of RNA was normalized against HuR content . Anti GFP - antibody was used for immunoprecipitation control ( C ) . Values are mean + / - s . e . m . and , n = 3Effect of GW4869 , the inhibitor of exosome mediated miRNA export , on exosomal miRNA content released by LPS stimulated cells . The level of miRNAs , let - 7a and miR - 155 were measured in exosomes ( EV ) released from LPS activated cell after GW4869 treatment . Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit . Values are mean + / - s . e . m . and , n = 3Effect of GW4869 on cellular cytokine levels in LPS treated cells . The effect of GW4869 on cellular expression of pro - inflammatory cytokines IL - 1β and TNF - α in LPS - activated macrophages were measured and normalized against 18S rRNA . Values in naive RAW 264 . 7 cells without GW4869 and LPS treatment were set as unit . Values are mean + / - s . e . m . and , n = 3Effect of siRNA - mediated downregulation of protein HuR on cellular expression of pro - inflammatory cytokines . In the left panel of F , effect of siRNA treatment on cellular cytokine mRNA levels in LPS - stimulated RAW 264 . 7 cells is shown . Relative levels measured against 18S rRNA are plotted . The effect of HuR depletion on cellular miR - 155 content has been measured and relative values normalized against U6 snRNA are plotted ( right panel , F ) . The amount of let - 7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 ( G ) . Values are mean + / - s . e . m . and , n = 3Effect of HuR expression on inflammatory responses in macrophage cells . Effect of ectopic expression of HuR on exosomal let - 7a content in naive macrophage cells ( H , right panel ) . HuR over expression was checked by western blot ( H , left panel ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n = 3Effect of HuR expression on inflammatory responses in macrophage cells . The effect of HuR expression on production of pro - inflammatory cytokine TNF - α in RAW 264 . 7 cells was measured . mRNA and protein level of TNF - α was quantified . Values obtained with pCINeo expression and without LPS treatment was taken as unit ( I ) . Transfection was done either with pCIneo ( control plasmid ) of HA - HuR expression construct and their effect on cellular pro - inflammatory cytokine mRNA levels in RAW64 . 7 cells was determined . Values in control set were taken as unit . Values are mean + / - s . e . m . and , n = 3Effect of HA - HuR expression on p38 mediated activation of downstream signalling events . The Levels of p - p38 and p - MSK1 have been monitored by western blotting done with cell extracts prepared from HA - HuR and control plasmid transfected , untreated and LPS treated RAW 264 . 7 cells . β - Actin blot was used as loading control . Downregulation of HuR in Ld - infected macrophages . HuR levels were monitored against time in Ld - infected primary macrophage cells ( PEC ) from BALB / c mice ( K , left upper panel ) as well as in Ld - infected RAW 264 . 7 cells ( K , upper right panel ) . HuR levels were also monitored against time in L . major - infected RAW 264 . 7 cells ( K , lower left panel ) and L . mexicana - infected RAW 264 . 7 cells ( K , lower right panel ) . Downregulation of HuR in Ld - infected macrophages . Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld - infected RAW 264 . 7 cells . In parallel assays levels of pro - inflammatory cytokine TNF - α was measured in Ld - infected cells either with no treatment or pre - treated with MG132 ( L ) . Values are mean + / - s . e . m . and , n = 3 . Downregulation of HuR in Ld - infected macrophages . The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined . Input samples were monitored for HuR downregulation by western blot . Presence of Alix ( marker for early endosomes ) and calnexin ( marker protein for ER ) were used to confirm separation of organelles during density gradient fractionation . Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection ( M upper panel ) . Densitometric analysis of individual fractions was done and the respective values were plotted ( M , lower panel ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5LPS induced miRNA export from mammalian macrophage cells. A schematic diagram of the experiments has been described in left panel of (A). Characterization and quantification of exosomes derived from Control and LPS-activated RAW 264.7 cells by Nanoparticle Tracking Analysis (NTA) done for isolated exosomes. Relative size distributions are shown (A, right panel).Levels of HuR and exosomal miRNA isolated from control and 24 hrs of LPS treated RAW 264.7 cells. RNA content was normalized against total exosomal proteins (B). β-Actin as loading control for HuR western blot.Binding of miRNAs with HuR after LPS exposure was measured. Amount of RNA was normalized against HuR content. Anti GFP-antibody was used for immunoprecipitation control (C). Values are mean+/- s.e.m. and, n=3Effect of GW4869, the inhibitor of exosome mediated miRNA export, on exosomal miRNA content released by LPS stimulated cells. The level of miRNAs, let-7a and miR-155 were measured in exosomes (EV) released from LPS activated cell after GW4869 treatment. Values for EVs from naïve macrophages treated without LPS without GW4869 treatment was set as unit. Values are mean+/- s.e.m. and, n=3Effect of GW4869 on cellular cytokine levels in LPS treated cells. The effect of GW4869 on cellular expression of pro-inflammatory cytokines IL-1β and TNF-α in LPS-activated macrophages were measured and normalized against 18S rRNA. Values in naive RAW 264.7 cells without GW4869 and LPS treatment were set as unit. Values are mean+/- s.e.m. and, n=3Effect of siRNA-mediated downregulation of protein HuR on cellular expression of pro-inflammatory cytokines. In the left panel of F, effect of siRNA treatment on cellular cytokine mRNA levels in LPS-stimulated RAW 264.7 cells is shown. Relative levels measured against 18S rRNA are plotted. The effect of HuR depletion on cellular miR-155 content has been measured and relative values normalized against U6 snRNA are plotted (right panel, F).The amount of let-7a bound to Ago2 after LPS stimulation in presence and absence of siHuR has been calculated and relative values has been normalized to immunoprecipitated Ago2 (G) .Values are mean+/- s.e.m. and, n=3Effect of HuR expression on inflammatory responses in macrophage cells. Effect of ectopic expression of HuR on exosomal let-7a content in naive macrophage cells (H, right panel). HuR over expression was checked by western blot (H, left panel). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3Effect of HuR expression on inflammatory responses in macrophage cells. The effect of HuR expression on production of pro-inflammatory cytokine TNF-α in RAW 264.7 cells was measured. mRNA and protein level of TNF-α was quantified. Values obtained with pCINeo expression and without LPS treatment was taken as unit (I). Transfection was done either with pCIneo (control plasmid) of HA-HuR expression construct and their effect on cellular pro-inflammatory cytokine mRNA levels in RAW64.7 cells was determined. Values in control set were taken as unit. Values are mean+/- s.e.m. and, n=3Effect of HA-HuR expression on p38 mediated activation of downstream signalling events. The Levels of p-p38 and p-MSK1 have been monitored by western blotting done with cell extracts prepared from HA-HuR and control plasmid transfected, untreated and LPS treated RAW 264.7 cells. β-Actin blot was used as loading control.Downregulation of HuR in Ld-infected macrophages. HuR levels were monitored against time in Ld-infected primary macrophage cells (PEC) from BALB/c mice (K, left upper panel) as well as in Ld-infected RAW 264.7 cells (K, upper right panel) .HuR levels were also monitored against time in L. major-infected RAW 264.7 cells (K, lower left panel) and L. mexicana-infected RAW 264.7 cells (K, lower right panel).Downregulation of HuR in Ld-infected macrophages. Effect of proteasomal inhibitor MG132 treatment on HuR protein levels in control and 6 hrs of Ld-infected RAW 264.7 cells. In parallel assays levels of pro-inflammatory cytokine TNF-α was measured in Ld-infected cells either with no treatment or pre-treated with MG132 (L). Values are mean+/- s.e.m. and, n=3.Downregulation of HuR in Ld-infected macrophages. The isotonic cell lysates prepared from 6hrs Ld infected cells were analysed on an OptiprepR density gradient and HuR levels in individual fractions were determined. Input samples were monitored for HuR downregulation by western blot. Presence of Alix (marker for early endosomes) and calnexin (marker protein for ER) were used to confirm separation of organelles during density gradient fractionation. Loss of HuR from ER associated fraction has been noted along with their reduction in total lysate after Ld infection (M upper panel). Densitometric analysis of individual fractions was done and the respective values were plotted (M, lower panel).


Figure 6effect of HA - HuR expression on PP2A protein levelseffect of HA - HuR expression on PP2A RNA levels were scored . Expression profiles of HuR , PP2A and miRNA let - 7a are connected to IL - 6 expression in RAW 264 . 7 cells . Data obtained from experiments described in previous figures are summed up to plot the changes in IL - 6 mRNA levels against time of LPS treatment along with changes in PP2A , miRNA let - 7a and HuR ( D ) . Change in expression of IL - 6 is connected to changes in let - 7a concentration . The mathematical equation fitting the curve are shown here . Y represents concentration of IL - 6 and X represents miRNA let - 7a concentration . A1 is the initial IL - 6 levels and A2 is the changed level . LogX0 is the mid - point of value 0 . 34 , a concentration of let - 7a where Il - 6 expression is reduced to halfvariations of IL - 6 expression with the altered level of PP2A ( black ) and HuR ( red ) . For both cases , the data were fitted to an equation , which is shown above . The parameter m denotes the expression level of PP2A / HuR which corresponds to the maximum IL - 6 expression ; w denotes the width of the distributions for both cases . For PP2A and HuR , the values of m were found to be 0 . 6 and 0 . 9 respectively and A is a constant ( F ) .

O B-PANEL_START O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6effect of HA-HuR expression on PP2A protein levelseffect of HA-HuR expression on PP2A RNA levels were scored.Expression profiles of HuR, PP2A and miRNA let-7a are connected to IL-6 expression in RAW 264.7 cells. Data obtained from experiments described in previous figures are summed up to plot the changes in IL-6 mRNA levels against time of LPS treatment along with changes in PP2A, miRNA let-7a and HuR (D).Change in expression of IL-6 is connected to changes in let-7a concentration. The mathematical equation fitting the curve are shown here. Y represents concentration of IL-6 and X represents miRNA let-7a concentration. A1 is the initial IL-6 levels and A2 is the changed level. LogX0 is the mid-point of value 0.34, a concentration of let-7a where Il-6 expression is reduced to halfvariations of IL-6 expression with the altered level of PP2A (black) and HuR (red). For both cases, the data were fitted to an equation, which is shown above. The parameter m denotes the expression level of PP2A/HuR which corresponds to the maximum IL-6 expression; w denotes the width of the distributions for both cases. For PP2A and HuR, the values of m were found to be 0.6 and 0.9 respectively and A is a constant (F).


Figure 7Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . HA - HuR was immunostained with anti - HA antibody and detected by secondary antibody tagged with AlexaR564 ( A ) . Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on internalized parasite number HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells . In the zoomed part of the merged picture are used to show the internalized parasite . Internalized parasites present in HA - HuR positive cells were counted and compared against untransfected cells without HA - HuR expression ( B ) . Effect of HA - HuR expression on Ld infection of RAW 264 . 7 cells . Effect of HuR expression on cytokine expression in RAW 264 . 7 cells . HA - HuR was transfected to RAW 264 . 7 cells and infection was given for 24 hrs at a host to parasite 1 : 10 ratio . Levels of expression of different cytokines are measured in control or HA - HuR expressing RAW 264 . 7 cells infected with Ld ( C ) . Scale bar 20 µm . Values are mean + / - s . e . m . and , n = 3 . Effect of Ld infection on the HuR expression in mouse liver . BALB / c mice of 6 weeks of age were infected with Ld via cardiac puncture . Animals were sacrificed after subsequent days ; protein was extracted from liver tissue followed by western blot analysis for HuR . β - Actin was used as loading control ( D , left panel ) . Each lane have designated individual animal . Band intensities were determined by ImageJ . In each case , HuR level was normalized by its respective β - Actin bands ( D , right panel mean + / - s . e . m . , n = 5 ) . Animals were injected with GP63 containing liposome ( gp1 - 3 ) or empty liposome ( C1 - 2 ) and after 24 hrs of injection , HuR level was checked against β - Actin in liver tissue . β - Actin was used as loading control ( E ) . Effect of expression of HA - HuR on Ld infection in mice liver . Scheme for animal experiment done with HA - HuR expression in liver is shown . Also the HA - HuR level was detected in the liver tissue by western blot analysis using anti - HA antibody ( F ) . Effect of expression of HA - HuR on Ld infection in mice liver . Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA . Values are mean + / - s . e . m . and , n = 5 ( G ) . Effect of expression of HA - HuR on Ld infection in mice liver . TNF - α ( Values are mean + / - s . e . m . and , n = 5 ) and IL - 10 ( Values are mean + / - s . e . m . and , n = 4 ) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted ( H and I , left panels ) . Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count , TNF - α and IL - 10 mRNA levels ( G mean + / - s . e . m . , ( n = 5 ) , H mean + / - s . e . m . , ( n = 6 ) and I mean + / - s . e . m . , ( n = 4 ) , right panel ) . Effect of HA - HuR plasmid tail - vein injection on mice spleen . Levels of HA - HuR were undetectable in mice spleen after expression plasmid was injected . Sample from one liver tissue of HA - HuR injected group was used as a positive control of HA - HuR expression in moue liver after tail vein injection of HuR encoding plasmid ( J ) . Effect of HA - HuR plasmid tail - vein injection on mice spleen . Simultaneously , TNF - α mRNA levels were checked in spleen total RNA ( K ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number and cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. HA-HuR was immunostained with anti-HA antibody and detected by secondary antibody tagged with AlexaR564 (A).Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on internalized parasite number HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Ld was stained with CFSE dye and parasite internalization was detected by counting the CFSE positive structures inside the cells. In the zoomed part of the merged picture are used to show the internalized parasite. Internalized parasites present in HA-HuR positive cells were counted and compared against untransfected cells without HA-HuR expression (B).Effect of HA-HuR expression on Ld infection of RAW 264.7 cells. Effect of HuR expression on cytokine expression in RAW 264.7 cells. HA-HuR was transfected to RAW 264.7 cells and infection was given for 24 hrs at a host to parasite 1:10 ratio. Levels of expression of different cytokines are measured in control or HA-HuR expressing RAW 264.7 cells infected with Ld (C). Scale bar 20 µm. Values are mean+/- s.e.m. and, n=3.Effect of Ld infection on the HuR expression in mouse liver. BALB/c mice of 6 weeks of age were infected with Ld via cardiac puncture. Animals were sacrificed after subsequent days; protein was extracted from liver tissue followed by western blot analysis for HuR. β- Actin was used as loading control (D, left panel). Each lane have designated individual animal. Band intensities were determined by ImageJ. In each case, HuR level was normalized by its respective β- Actin bands (D, right panel mean +/- s.e.m., n=5).Animals were injected with GP63 containing liposome (gp1-3) or empty liposome (C1-2) and after 24 hrs of injection, HuR level was checked against β-Actin in liver tissue. β-Actin was used as loading control (E).Effect of expression of HA-HuR on Ld infection in mice liver. Scheme for animal experiment done with HA-HuR expression in liver is shown. Also the HA-HuR level was detected in the liver tissue by western blot analysis using anti-HA antibody (F).Effect of expression of HA-HuR on Ld infection in mice liver. Parasite load in liver tissues were estimated by measuring Ld DNA in infected tissue using specific primer for Ld minicircle kDNA. Values are mean+/- s.e.m. and, n=5 (G).Effect of expression of HA-HuR on Ld infection in mice liver. TNF-α (Values are mean+/- s.e.m. and, n=5) and IL-10 (Values are mean+/- s.e.m. and, n=4) mRNA levels were checked from RNA isolated from liver tissues and 18S rRNA normalized Ct values were plotted (H and I, left panels). Relative fold change was calculated by ΔΔCt method and the values plotted for Ld count, TNF-α and IL-10 mRNA levels (G mean +/- s.e.m., (n=5), H mean +/- s.e.m., (n=6) and I mean +/- s.e.m., (n=4), right panel).Effect of HA-HuR plasmid tail-vein injection on mice spleen. Levels of HA-HuR were undetectable in mice spleen after expression plasmid was injected. Sample from one liver tissue of HA-HuR injected group was used as a positive control of HA-HuR expression in moue liver after tail vein injection of HuR encoding plasmid (J).Effect of HA-HuR plasmid tail-vein injection on mice spleen. Simultaneously, TNF-α mRNA levels were checked in spleen total RNA (K).


Figure 8Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on cellular cytokine level RAW 264 . 7 cells were infected with Antimony sensitive ( LdS - Ag83 ) and antimony resistant ( LdR - BHU569 ) form of Ld for 24 hours along with uninfected cells kept as control . IL - 10 ( A ) and TNF - α ( B ) were measured at mRNA level by qRT - PCR after infection ( mean + / - s . e . m . and , n = 4 ) . Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on on phospho - ERK level . ( D ) . Densitometry analysis was done for p - ERK level by ImageJ and relative values were plotted ( C ) . β - Actin was used as loading control Values are mean + / - s . e . m . and , n = 3 . Effect of antimonial drug resistant and sensitive forms of the Ld ( LdR and LdS ) on phospho - ERK and MDR1 level . p - ERK , ERK and MDR1 level were also measured by western blot ( D ) . Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A . RAW 264 . 7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot ( left panel ) . PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection ( right panel ) . β - Actin was used as loading control . Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell . RAW 264 . 7 cells were transfected with HA - HuR or pCIneo followed by 2 hrs of pretreatment with OA . These cells were then infected with LdR for 24 hrs . Pro - inflammatory cytokines TNF - α ( right panel ; mean + / - s . e . m . , n = 3 ) and IL - 1β ( left panel ; mean + / - s . e . m . and , n = 4 ) were measured at mRNA level and relative values are plotted . Values without OA treatment was set as control . Effect of HuR over expression on LdR infection in mice liver . Pro - inflammatory cytokines TNF - α ( left panel ) and IL - 1β ( right panel ) level were measured at mRNA level in liver ( H ) . Effect of HuR over expression on LdR infection in mice liver . Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld ( I ) . Values are mean + / - s . e . m . and , n = 6 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on cellular cytokine level RAW 264.7 cells were infected with Antimony sensitive (LdS-Ag83) and antimony resistant (LdR-BHU569) form of Ld for 24 hours along with uninfected cells kept as control. IL-10 (A) and TNF-α (B) were measured at mRNA level by qRT-PCR after infection (mean+/- s.e.m. and, n=4).Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on on phospho-ERK level. (D). Densitometry analysis was done for p-ERK level by ImageJ and relative values were plotted (C). β-Actin was used as loading control Values are mean+/- s.e.m. and, n=3.Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on phospho-ERK and MDR1 level. p-ERK, ERK and MDR1 level were also measured by western blot (D).Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A. RAW 264.7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot (left panel). PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection (right panel). β-Actin was used as loading control.Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.Effect of HuR over expression on LdR infection in mice liver. Pro-inflammatory cytokines TNF-α (left panel) and IL-1β (right panel) level were measured at mRNA level in liver (H).Effect of HuR over expression on LdR infection in mice liver. Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld (I). Values are mean+/- s.e.m. and, n=6).


Figure 1 ( A ) Gene set enrichment analysis ( GSEA ) of differentially expressed genes in colorectal cancer from the GEO database ( GDS4382 ) . ES , enrichment score ; NES , normalized enrichment score . n = 3 biological samples . ( B ) Kaplan - Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset ( n = 590 samples , P = 0 . 016 , Log - rank ( Mantel - Cox ) test ) . " Normal " represents the patients with unaltered expression of iron homoeostasis - related genes , including LTF , CP , FTH1 , SLC30A1 and TFRC ; " Dysfunctional iron homoeostasis " represents the patients with altered expression of iron homoeostasis - related genes . ( C ) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues ( Left ) , the relative protein level of IREB2 was assessed by ImageJ software ( Right ) ( n = 8 human samples , * P = 0 . 0114 , two - tailed paired Student ' s t - test ) . ( D ) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue , and was subsequently analyzed by anti - ubiquitin antibody for the assessment of ubiquitination . ( E ) Mass spectrum analysis of IREB2 - associated proteins . Mock or IREB2 - FLAG was transfected into HEK293T cells and FLAG - tagged proteins were enriched by anti - FLAG M2 beads and incubated with lysates from normal mice colon tissues . Five matched peptides corresponding to OTUD1 were shown on the right panel . ( F ) In vivo ubiquitination assay of IREB2 . HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti - FLAG antibody followed by western blot analysis . ( G ) Half - life analysis of IREB2 in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . Cells were treated with 100 μg / ml cycloheximide ( CHX ) and collected at the indicated times for western blot analysis ( Up ) , the relative protein level of IREB2 was assessed by ImageJ software ( Down ) ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0049 , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . ( H ) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC ( 50 μM ) and Hemin ( 100 μM ) treatment . MFI , mean fluorescence intensity . Control uses isotype - matched control antibody ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0010 , * * * P = 0 . 0008 , two - tailed unpaired Student ' s t - test ) . ( I ) Flow cytometric analysis of TFRC expression in wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells with DFO ( 100 μM ) treatment . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Gene set enrichment analysis (GSEA) of differentially expressed genes in colorectal cancer from the GEO database (GDS4382). ES, enrichment score; NES, normalized enrichment score. n = 3 biological samples.(B) Kaplan-Meier survival curves for colon cancer patients with or without dysfunction of iron homeostasis in the GSE dataset (n = 590 samples, P = 0.016, Log-rank (Mantel-Cox) test). "Normal" represents the patients with unaltered expression of iron homoeostasis-related genes, including LTF, CP, FTH1, SLC30A1 and TFRC; "Dysfunctional iron homoeostasis" represents the patients with altered expression of iron homoeostasis-related genes. (C) Immunoblot analysis of protein levels of IREB2 in colorectal adenocarcinoma and its matched adjacent normal tissues (Left), the relative protein level of IREB2 was assessed by ImageJ software (Right) (n = 8 human samples, *P = 0.0114, two-tailed paired Student's t-test).  (D) Endogenous IREB2 was immunoprecipitated from the lysates of primary colorectal adenocarcinoma and its matched adjacent normal tissue, and was subsequently analyzed by anti-ubiquitin antibody for the assessment of ubiquitination.  (E) Mass spectrum analysis of IREB2-associated proteins. Mock or IREB2-FLAG was transfected into HEK293T cells and FLAG-tagged proteins were enriched by anti-FLAG M2 beads and incubated with lysates from normal mice colon tissues. Five matched peptides corresponding to OTUD1 were shown on the right panel.  (F) In vivo ubiquitination assay of IREB2. HEK293T cells were transfected with indicated plasmids and subjected to immunoprecipitation with anti-FLAG antibody followed by western blot analysis.  (G) Half-life analysis of IREB2 in wild-type (WT) and OTUD1─/─ NCM460 cells. Cells were treated with 100 μg/ml cycloheximide (CHX) and collected at the indicated times for western blot analysis (Up), the relative protein level of IREB2 was assessed by ImageJ software (Down) (n = 3 biological replicates, mean ± s.e.m., **P = 0.0049, ***P < 0.0001, two-tailed unpaired Student's t-test).  (H) Flow cytometric analysis of TFRC expression in mock and OTUD1 expressing CT26 cells with AFC (50 μM) and Hemin (100 μM) treatment. MFI, mean fluorescence intensity. Control uses isotype-matched control antibody (n = 4 biological replicates, mean ± s.e.m., **P = 0.0010, ***P = 0.0008, two-tailed unpaired Student's t-test).  (I) Flow cytometric analysis of TFRC expression in wild-type (WT) and OTUD1─/─ NCM460 cells with DFO (100 μM) treatment. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., ***P = 0.0001, two-tailed unpaired Student's t-test).


Figure 2 ( A ) RT - qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues ( n = 101 human samples , * * * P < 0 . 001 , two - tailed paired Student ' s t - test ) ( B ) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer ( n = 101 human samples , two - tailed Pearson correlation analysis , * * * * P < 0 . 0001 ) . ( C ) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues ( n = 4 human samples ) , the protein levels relative to GAPDH are marked . ( D ) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue ( The scale bars represent 1000 μm ) . ( E ) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05 - 118e . ( F ) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a ( The scale bars represent 1000 μm ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (A) RT-qPCR analysis of OTUD1 mRNA levels in colon tumors and matched adjacent normal tissues (n = 101 human samples, ***P < 0.001, two-tailed paired Student's t-test)  (B) Correlation of mRNA expression of TFRC and OTUD1 in colorectal cancer (n = 101 human samples, two-tailed Pearson correlation analysis, ****P < 0.0001).  (C) Immunoblot analysis of protein levels of OTUD1 and TFRC in colorectal adenocarcinoma and its matched adjacent normal tissues (n = 4 human samples), the protein levels relative to GAPDH are marked.  (D) Immunohistochemical staining of OTUD1 in colorectal adenocarcinoma and its matched adjacent normal tissue (The scale bars represent 1000 μm).  (E) Analysis of OTUD1 protein levels in colorectal adenocarcinoma patients with different clinical stages from tissue microarray CoI05-118e. (F) Representative immunohistochemistry staining pictures of OTUD1 protein levels in normal tissue and different colon diseases from tissue microarray CO809a (The scale bars represent 1000 μm).


Figure 3 Intracellular iron concentration was measured in CT26 cells stably expressing mock , OTUD1 or OTUD1C320S ( n = 2 biological replicates ) Intracellular iron concentration was measured in wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( C ) The protein levels of IREB2 , TFRC and OTUD1 were assessed by western blot in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice . ( D ) Intracellular iron concentration was measured in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice with supplementation of ferrous gluconate ( n = 3 biological replicates , mean ± s . e . m . , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) . Mean corpuscular volume ( MCV ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0472 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . mean corpuscular hemoglobin ( MCH ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0048 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . mean corpuscular hemoglobin concentration ( MCHC ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0068 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . , red boold cells ( RBCs ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0049 , two - tailed unpaired Student ' s t - test ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times . hemoglobin ( HB ) ( n = 5 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P = 0 . 0036 , * * * P = 0 . 0002 , two - tailed unpaired Student ' s t - test ) ( I ) in wild - type ( WT ) and Otud1 ─ / ─ mice with or without low - iron diets for indicated times .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 Intracellular iron concentration was measured in CT26 cells stably expressing mock, OTUD1 or OTUD1C320S (n = 2 biological replicates)  Intracellular iron concentration was measured in wild-type (WT) or OTUD1─/─ NCM460 cells (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test)  (C) The protein levels of IREB2, TFRC and OTUD1 were assessed by western blot in colon tissues from wild-type (WT) or Otud1─/─ mice.  (D) Intracellular iron concentration was measured in colon tissues from wild-type (WT) or Otud1─/─ mice with supplementation of ferrous gluconate (n = 3 biological replicates, mean ± s.e.m., ***P = 0.0007, two-tailed unpaired Student's t-test).  Mean corpuscular volume (MCV) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0472, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin (MCH) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0048, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  mean corpuscular hemoglobin concentration (MCHC) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0068, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  , red boold cells (RBCs) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0049, two-tailed unpaired Student's t-test) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.  hemoglobin (HB) (n = 5 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P = 0.0036, ***P = 0.0002, two-tailed unpaired Student's t-test) (I) in wild-type (WT) and Otud1─/─ mice with or without low-iron diets for indicated times.


Figure 4 ( A - B ) Macroscopic evaluation ( A ) and tumor volume ( mock , n = 7 biological replicates ; OTUD1 , n = 9 biological replicates ; OTUD1C320S , n = 9 biological replicates ; mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) ( B ) of mock , OTUD1 and OTUD1C320S expressing CT26 tumors in BALB / c mice . Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . tumor volume ( n = 6 biological replicates , mean ± s . e . m . , * * P < 0 . 01 , * * * P = 0 . 0007 , two - tailed unpaired Student ' s t - test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . survival analysis ( n = 5 biological replicates , * * P = 0 . 0021 , Log - rank ( Mantel - Cox ) test ) of mock and OTUD1 expressing LLC tumors in C57BL / 6 mice . ( F ) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti - IREB2 antibody and anti - TFRC antibody . ( G ) Flow cytometric analysis of TFRC expression in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors . MFI , mean fluorescence intensity ( mean ± s . e . m . , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( H ) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors . MFI , mean fluorescence intensity . ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0011 , two - tailed unpaired Student ' s t - test ) . ( I ) Iron concentration of mock , OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry ( n = 3 biological replicates , mean ± s . e . m . , * P = 0 . 0237 , * * P = 0 . 0098 , two - tailed unpaired Student ' s t - test ) . ( J ) Analysis of tumor infiltrating CD8 + T cells or CD4 + T cells in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) and OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors in BALB / c mice ( mean ± s . e . m . , * * P < 0 . 01 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . Flow cytometric analysis of CD8 + T cells , CD4 + T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P = 0 . 0003 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors . PD - 1 expression in T cells ( n = 6 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) isolated from mock and OTUD1 overexpressing LLC tumors . MFI , mean fluorescence intensity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (A-B) Macroscopic evaluation (A) and tumor volume (mock, n = 7 biological replicates; OTUD1, n = 9 biological replicates; OTUD1C320S, n = 9 biological replicates; mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) (B) of mock, OTUD1 and OTUD1C320S expressing CT26 tumors in BALB/c mice.  Macroscopic evaluation of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  tumor volume (n=6 biological replicates, mean ± s.e.m., **P < 0.01, ***P = 0.0007, two-tailed unpaired Student's t-test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  survival analysis (n=5 biological replicates, **P = 0.0021, Log-rank (Mantel-Cox) test) of mock and OTUD1 expressing LLC tumors in C57BL/6 mice.  (F) Endogenous IREB2 and TFRC protein levels in murine CT26 tumors were measured by anti-IREB2 antibody and anti-TFRC antibody.  (G) Flow cytometric analysis of TFRC expression in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors. MFI, mean fluorescence intensity (mean ± s.e.m., **P < 0.01, two-tailed unpaired Student's t-test).  (H) Flow cytometric analysis of TFRC expression in mock or OTUD1 expressing LLC tumors. MFI, mean fluorescence intensity. (n = 5 biological replicates, mean ± s.e.m., **P = 0.0011, two-tailed unpaired Student's t-test).  (I) Iron concentration of mock, OTUD1 and OTUD1C320S tumors was assessed by Inductively Coupled Plasma Mass Spectrometry (n = 3 biological replicates, mean ± s.e.m., *P = 0.0237, **P = 0.0098, two-tailed unpaired Student's t-test).  (J)Analysis of tumor infiltrating CD8+ T cells or CD4+ T cells in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) and OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors in BALB/c mice (mean ± s.e.m., **P < 0.01, ***P < 0.001, two-tailed unpaired Student's t-test).  Flow cytometric analysis of CD8+ T cells, CD4+ T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P = 0.0003, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors.  PD-1 expression in T cells (n = 6 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), ***P < 0.0001, two-tailed unpaired Student's t-test) isolated from mock and OTUD1 overexpressing LLC tumors. MFI, mean fluorescence intensity.


Figure 5 A ) Quantitative proteomics study of wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells . The expression of TFRC and IREB2 were highlighted with red color . Intensity indicates label - free quantification ( LFQ ) intensity of proteins in mass spectrometry . ( B ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * P < 0 . 05 , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( C ) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer - 1 ( 10 μM ) , Z - VAD ( 25 μM ) and nec - 1 ( 20 μM ) . Cell viability was measured by PI staining n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * P = 0 . 0119 , * * P = 0 . 0015 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . ( D ) CT26 cells stably expressing mock , OTUD1 or OTUD1C320S were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) , and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , ns , not significant ( P > 0 . 05 ) , * * P < 0 . 01 , two - tailed unpaired Student ' s t - test ) . ( E ) Wild - type ( WT ) and OTUD1 ─ / ─ NCM460 cells were treated with Erastin ( 10 μM ) and RSL3 ( 5 μM ) for 24 hours and cell viability was measured by PI staining ( n = 3 biological replicates , mean ± s . e . m . , * * P = 0 . 0082 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . F ) Intracellular ROS levels in mock , OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin ( 10 μM ) , AFC ( 50 μM ) or H2O2 ( 100 μM ) were detected by DCFDA staining . MFI , mean fluorescence intensity ( n = 2 biological replicates ) . ( G ) Intracellular ROS levels in mock ( n = 4 biological replicates ) , OTUD1 ( n = 6 biological replicates ) or OTUD1C320S ( n = 5 biological replicates ) expressing CT26 tumors ( mean ± s . e . m . , * P = 0 . 0189 , * * P = 0 . 0086 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity . ( I ) Tumor volume ( mock , n = 4 biological replicates ; mock + NP - VE , n = 6 biological replicates ; OTUD1 , n = 4 biological replicates ; OTUD1 + NP - VE , n = 6 biological replicates Flow cytometric analysis of DCFDA ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE . MFI , mean fluorescence intensity . Flow cytometric analysis of the percentage of tumor infiltrating CD8 + T cells ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0433 , * * * P = 0 . 0001 , two - tailed unpaired Student ' s t - test ) of mock or OTUD1 expressing CT26 tumors treated with or without NP - VE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A) Quantitative proteomics study of wild-type (WT) and OTUD1─/─ NCM460 cells. The expression of TFRC and IREB2 were highlighted with red color. Intensity indicates label-free quantification (LFQ) intensity of proteins in mass spectrometry.  (B) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with indicated concentration of H2O2 for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., *P < 0.05, **P < 0.01, two-tailed unpaired Student's t-test).  (C) CT26 cells stably expressing mock or OTUD1 were treated with 200 μM H2O2 together with various cell death inhibitors containing Fer-1 (10 μM), Z-VAD (25 μM) and nec-1 (20 μM). Cell viability was measured by PI staining n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), *P = 0.0119, **P = 0.0015, ***P < 0.001, two-tailed unpaired Student's t-test).  (D) CT26 cells stably expressing mock, OTUD1 or OTUD1C320S were treated with Erastin (10 μM) and RSL3 (5 μM), and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., ns, not significant (P > 0.05), **P < 0.01, two-tailed unpaired Student's t-test).  (E) Wild-type (WT) and OTUD1─/─ NCM460 cells were treated with Erastin (10 μM) and RSL3 (5 μM) for 24 hours and cell viability was measured by PI staining (n = 3 biological replicates, mean ± s.e.m., **P = 0.0082, ***P < 0.001, two-tailed unpaired Student's t-test).  F) Intracellular ROS levels in mock, OTUD1 and OTUD1C320S overexpressing CT26 cells treated with Erastin (10 μM), AFC (50 μM) or H2O2 (100 μM) were detected by DCFDA staining. MFI, mean fluorescence intensity (n = 2 biological replicates).  (G) Intracellular ROS levels in mock (n = 4 biological replicates), OTUD1 (n = 6 biological replicates) or OTUD1C320S (n = 5 biological replicates) expressing CT26 tumors (mean ± s.e.m., *P = 0.0189, **P = 0.0086, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.  (I) Tumor volume (mock, n = 4 biological replicates; mock + NP-VE, n = 6 biological replicates; OTUD1, n = 4 biological replicates; OTUD1 + NP-VE, n = 6 biological replicates  Flow cytometric analysis of DCFDA (n = 4 biological replicates, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE. MFI, mean fluorescence intensity.  Flow cytometric analysis of the percentage of tumor infiltrating CD8+ T cells (n = 4 biological replicates, mean ± s.e.m., *P = 0.0433, ***P = 0.0001, two-tailed unpaired Student's t-test) of mock or OTUD1 expressing CT26 tumors treated with or without NP-VE.


Figure 6 CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . wild - type ( WT ) or OTUD1 ─ / ─ NCM460 cells were treated with 5 μM RSL3 , and medium was collected to measure ATP level ( n = 4 biological replicates , mean ± s . e . m . , * * * P < 0 . 0001 , two - tailed unpaired Student ' s t - test ) . ( C ) Volcano plots of genes differentially expressed in tumor interstitial fluid ( TIF ) of mock and OTUD1 expressing CT26 tumors . Left , example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid ; right , example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid . ( D ) Immunoblot analysis of protein levels of HMGB1 , HSP70 and HSP90 in tumor interstitial fluid of mock , OTUD1 and OTUD1C320S expressing CT26 tumors . ( E ) ATP level in tumor interstitial fluid ( TIF ) of mock or OTUD1 overexpressing CT26 cells ( n = 5 mice , mean ± s . e . m . , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test ) . ( F and G ) BALB / c mice were immunized with 1 × 106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2 × 106 wild - type CT26 cells in the right flank 10 days later . Macroscopic evaluation ( F ) and tumor volume ( n = 7 mice , mean ± s . e . m . , * * P = 0 . 0053 , two - tailed unpaired Student ' s t - test ) ( G ) of tumors in the right flank were shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 CT26 cells stably expressing mock or OTUD1 were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).  wild-type (WT) or OTUD1─/─ NCM460 cells were treated with 5 μM RSL3, and medium was collected to measure ATP level (n = 4 biological replicates, mean ± s.e.m., ***P < 0.0001, two-tailed unpaired Student's t-test).  (C) Volcano plots of genes differentially expressed in tumor interstitial fluid (TIF) of mock and OTUD1 expressing CT26 tumors. Left, example of genes downregulated in OTUD1 overexpressing tumor interstitial fluid; right, example of genes upregulated in OTUD1 overexpressing tumor interstitial fluid.  (D) Immunoblot analysis of protein levels of HMGB1, HSP70 and HSP90 in tumor interstitial fluid of mock, OTUD1 and OTUD1C320S expressing CT26 tumors.  (E) ATP level in tumor interstitial fluid (TIF) of mock or OTUD1 overexpressing CT26 cells (n = 5 mice, mean ± s.e.m., ***P < 0.001, two-tailed unpaired Student's t-test).  (F and G) BALB/c mice were immunized with 1×106 OTUD1 expressing or mock CT26 cells in the left flank and challenged with 2×106 wild-type CT26 cells in the right flank 10 days later. Macroscopic evaluation (F) and tumor volume (n=7 mice, mean ± s.e.m., **P = 0.0053, two-tailed unpaired Student's t-test) (G) of tumors in the right flank were shown.


Figure 7 ( A ) Representative pictures of colon tumors in AOM / DSS - induced Colitis - Associated Cancer ( CAC ) model ( n = 6 biological replicates ) . ( B and C ) The tumor load ( B ) or tumor number ( C ) in the whole colon was measured ( n = 12 biological replicates , mean ± s . e . m . , * P = 0 . 0431 , * * P = 0 . 003 , two - tailed unpaired Student ' s t - test ) . ( D ) Representative H & E ( Hematoxylin - eosin ) staining pictures of colon tumors ( The scale bars represent 1000 μm ) . ( E ) Flow cytometric analysis of TFRC expression in intestinal epithelial cells ( IECs ) from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS . MFI , mean fluorescence intensity . ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0062 , two - tailed unpaired Student ' s t - test ) . ( F ) GSEA of genes expressed in colon tissues from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 3 biological replicates ) . ES , enrichment score ; NES , normalized enrichment score . ( G ) Representative immunohistochemistry staining pictures of CD3 + T cells in colon tumors ( The scale bars represent 400 μm ) . ( H ) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells ( LPMC ) isolated from wild - type ( WT ) and Otud1 ─ / ─ mice treated with AOM / DSS ( n = 5 biological replicates , mean ± s . e . m . , * * P = 0 . 0025 , * * * P = 0 . 0005 , two - tailed unpaired Student ' s t - test ) . ( I ) Intracellular ROS levels in colon tissues from wild - type ( WT ) or Otud1 ─ / ─ mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * * P = 0 . 0072 , two - tailed unpaired Student ' s t - test ) were detected by DCFDA staining . MFI , mean fluorescence intensity . ( J ) ATP level in tumor interstitial fluid ( TIF ) of mice treated with AOM / DSS ( n = 4 biological replicates , mean ± s . e . m . , * P = 0 . 0292 , two - tailed unpaired Student ' s t - test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 (A) Representative pictures of colon tumors in AOM/DSS-induced Colitis-Associated Cancer (CAC) model (n = 6 biological replicates).  (B and C) The tumor load (B) or tumor number (C) in the whole colon was measured (n = 12 biological replicates, mean ± s.e.m., *P = 0.0431, **P = 0.003, two-tailed unpaired Student's t-test).  (D) Representative H&E (Hematoxylin-eosin) staining pictures of colon tumors (The scale bars represent 1000 μm).  (E) Flow cytometric analysis of TFRC expression in intestinal epithelial cells (IECs) from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS. MFI, mean fluorescence intensity. (n = 4 biological replicates, mean ± s.e.m., **P = 0.0062, two-tailed unpaired Student's t-test).  (F) GSEA of genes expressed in colon tissues from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 3 biological replicates). ES, enrichment score; NES, normalized enrichment score.  (G) Representative immunohistochemistry staining pictures of CD3+ T cells in colon tumors (The scale bars represent 400 μm ).  (H) Flow cytometric analysis of various T cell subsets in lamina propria mononuclear cells (LPMC) isolated from wild-type (WT) and Otud1─/─ mice treated with AOM/DSS (n = 5 biological replicates, mean ± s.e.m., **P = 0.0025, ***P = 0.0005, two-tailed unpaired Student's t-test).  (I) Intracellular ROS levels in colon tissues from wild-type (WT) or Otud1─/─ mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., **P = 0.0072, two-tailed unpaired Student's t-test) were detected by DCFDA staining. MFI, mean fluorescence intensity.  (J) ATP level in tumor interstitial fluid (TIF) of mice treated with AOM/DSS (n = 4 biological replicates, mean ± s.e.m., *P = 0.0292, two-tailed unpaired Student's t-test).


Figure 1B . Semi - quantitative PCR of miR - 132 and negative control miRNAs in the hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected animals ( aCSF and scramble ) at six months of age . Sample size , n = 9 per group . C . FISH of miR - 132 and negative control miR - 124 in the hippocampus of ant - 132 - and scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm . D . Semi - quantitative PCR of miR - 132 and miR - 212 in the hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to animals injected with a negative control oligonucleotide ( Ctr ) . Sample size , n = 6 per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group.C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm.D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group.


Figure 2A . Amyloid staining ( 6E10 ) combined with miR - 132FISH in ant - 132 - and scramble - injected mice at six months of age . Scale bar , 500 μm . B . Quantification of amyloid burden in hippocampus and cortex of ant - 132 - injected and control animals ( see Materials and Methods ) . Sample size , n = 4 per group . C , D . ELISA of soluble ( TBS ) and insoluble ( Formic acid ) Aβ40 and Aβ42 levels in the hippocampus of ant - 132 - ( C ) and miR - 132 - injected ( D ) animals at six and three months of age , respectively . Sample size , n = 6 per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group.C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.


Figure 3Western blot analysis of total TAU and pTAU ( AT8 , AT270 ) levels upon miR - 132 knockdown ( A ) or overexpression ( B ) at six and three months of age , respectively . Sample size , n = 9 per group ( A ) and n = 6 per group ( B ) . Values were normalized to the respective control groups and presented as mean ± SEM . Student ' s t - test was used .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.


Figure 4B . Positive hits were validated by semi - quantitative qPCR in ant - 132 - injected mousehippocampus . Black line indicates mean of scramble control set to 1 . Dashed line indicates chosen threshold of 1 . 3 - fold . Sample size , n = 9 per group . Values were normalized to scramble - injected group and presented as mean ± SEM .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.