( D ) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells . Relative levels were estimated by real time qPCR , normalized with respect to U6 snRNA and plotted in the left panel . Levels of miR - 122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT - PCR and plotted in the right panel ( mean + / - s . e . m . , n = 3 ) .

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(D) Effect of HuR knockdown on cellular level of different miRNAs in starved Huh7 cells. Relative levels were estimated by real time qPCR, normalized with respect to U6 snRNA and plotted in the left panel. Levels of miR-122 in EVs of starved siHuR and siCon treated Huh7 cells were also measured by RT-PCR and plotted in the right panel (mean+/- s.e.m., n=3).


( E ) Level of eIF - 2a and its phosphorylated form , estimated by western blot using specific antibodies , in siCon or siHuR treated and starved Huh7 cell extracts . Western blot data of HuR confirm reduction of this protein upon siRNA treatment . CAT - 1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level . Values of siCon treated samples were taken as unit ( mean + / - s . e . m . , n = 3 ) . In the right panel the relative quantity of phosphorylated eIF - 2a measured by densitometry were plotted . Levels of p38 and phosphor - p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot .

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(E) Level of eIF-2a and its phosphorylated form, estimated by western blot using specific antibodies, in siCon or siHuR treated and starved Huh7 cell extracts. Western blot data of HuR confirm reduction of this protein upon siRNA treatment. CAT-1 and Aldolase mRNA levels were estimated by real time quantification and normalized against GAPDH level. Values of siCon treated samples were taken as unit (mean+/- s.e.m., n=3). In the right panel the relative quantity of phosphorylated eIF-2a measured by densitometry were plotted. Levels of p38 and phosphor-p38 levels in siCon and siHuR treated Starved Huh7 cells were detected by western blot.


( F ) Western blot analysis to detect the level of phosphorylated eIF - 2a and eIF - 4E - BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant - let - 7a or anti - miR - 122 oligos . miR - 122 inactivation was monitored by measuring cat - 1 level by qRT - PCR using GAPDH as control ( mean + / - s . e . m . , n = 3 ) . * denotes the phosphorylated eIF - 4E - BP1 . b - Actin was used as loading control . Relative levels of phosphorylated eIF - 2a were measured by densitometric estimation of three independent measurements .

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(F) Western blot analysis to detect the level of phosphorylated eIF-2a and eIF-4E-BP1 in lysates of Starved and HuR depleted Huh7 cells either transfected with ant-let-7a or anti-miR-122 oligos. miR-122 inactivation was monitored by measuring cat-1 level by qRT-PCR using GAPDH as control (mean+/- s.e.m., n=3). * denotes the phosphorylated eIF-4E-BP1. b-Actin was used as loading control. Relative levels of phosphorylated eIF-2a were measured by densitometric estimation of three independent measurements.


( A ) Northern blot of miR - 122 in Huh7 cells expressing the Control and HA - HuR expression plasmids . The U6 was used for loading control . Position of the 32P - labelled Oligos , blotted to the membrane used for Northern blotting , served as size markers are shown in the M lane .

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(A) Northern blot of miR-122 in Huh7 cells expressing the Control and HA-HuR expression plasmids. The U6 was used for loading control. Position of the 32P-labelled Oligos, blotted to the membrane used for Northern blotting, served as size markers are shown in the M lane.


( B ) Schematic representation of experiments done in Huh7 cells expressing HA - HuR ( bottom ) . For control , pCIneo transfected Huh7 cells were used . Relative expression of HuR in transfected cells were analysed by western blot ( top ) . b - actin blots were used as loading controls .

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(B) Schematic representation of experiments done in Huh7 cells expressing HA-HuR (bottom). For control, pCIneo transfected Huh7 cells were used. Relative expression of HuR in transfected cells were analysed by western blot (top). b-actin blots were used as loading controls.


( C ) Cellular and EV - associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA - HuR expressing vectors by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . Cellular miRNA levels were normalized against U6 snRNA .

(C) Cellular and EV-associated miRNA levels were measured in Huh7 cells either expressing the pCIneo or HA-HuR expressing vectors by qRT-PCR (mean+/- s.e.m., n=5). Cellular miRNA levels were normalized against U6 snRNA.


( E ) Effect of HA - HuR expression on miRNA levels on EV and non - EV fractions of the culture supernatant collected from control and HA - HuR encoding plasmid transfected Huh7 cells . For isolation of non - EV fraction , supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation ( left panel ) ( mean + / - s . e . m . , n = 3 ) . Effect of HuR expression on CD63 , Alix and HuR levels in EVs . Extracts from EVs isolated from control or HA - HuR expressing Huh7 cells were western blotted for respective proteins ( right panel ) .

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(E) Effect of HA-HuR expression on miRNA levels on EV and non-EV fractions of the culture supernatant collected from control and HA-HuR encoding plasmid transfected Huh7 cells. For isolation of non-EV fraction, supernatant obtained after removal of EVs by ultracentrifugation was used for RNA preparation (left panel) (mean+/- s.e.m., n=3). Effect of HuR expression on CD63, Alix and HuR levels in EVs. Extracts from EVs isolated from control or HA-HuR expressing Huh7 cells were western blotted for respective proteins (right panel).


( F ) Relative miR - 122 levels in CD63 positive affinity purified EVs isolated from HA - HuR and pCIneo transfected Huh7 cells were quantified by qRT - PCR ( mean + / - s . e . m . , n = 3 ) . The levels of miR - 122 were normalized against CD63 content of EVs .

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(F) Relative miR-122 levels in CD63 positive affinity purified EVs isolated from HA-HuR and pCIneo transfected Huh7 cells were quantified by qRT-PCR (mean+/- s.e.m., n=3). The levels of miR-122 were normalized against CD63 content of EVs.


( G ) Effect of GW4869 on EV associated content of three different miRNAs in HA - HuR expressing Huh7 cells quantified by qRT - PCR ( mean + / - s . e . m . , n = 5 ) . EVs from DMSO treated cells were used as control .

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(G) Effect of GW4869 on EV associated content of three different miRNAs in HA-HuR expressing Huh7 cells quantified by qRT-PCR (mean+/- s.e.m., n=5). EVs from DMSO treated cells were used as control.


( H ) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA - HuR or pCIneo respectively by running OptiPrepTM density gradient ( left panel ) and western blotting for CD63 ( right upper panel ) . Relative miR - 122 levels in fractions 1 - 2 and 8 - 9 were compared by qRT - PCR ( right lower panel ) .

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(H) A schematic representation of the experiment done to characterize EVs isolated from Huh7 cells expressing HA-HuR or pCIneo respectively by running OptiPrepTM density gradient (left panel) and western blotting for CD63 (right upper panel). Relative miR-122 levels in fractions 1-2 and 8-9 were compared by qRT-PCR (right lower panel).


( I ) Levels of apoptic cells in HA - HuR expressing Huh7 cells . Western Blot detection of apoptosis marker Cytochrome C was done ( top ) for EV associated and cellular fractions of HA - HuR and pCIneo ( control ) expressing Huh 7 cells respectively . Western blot for HA - HuR was done to analyse the expression of HA - HuR in EVs isolated from HA - HuR and pCIneo expressing Huh7 cells . CD63 and β - actin are used as loading control for EV associated fractions and cellular fractions respectively ( upper panels ) . TUNEL assay of HA - HuR and pCIneo expressing and non - transfected Huh 7 cells was performed and representative pictures of HA - HuR and pCIneo expressing cells along with picture of non - transfected but DNase treated ( positive control ) cells were compared ( lower panels ) . Scale bar 50 µM .

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(I) Levels of apoptic cells in HA-HuR expressing Huh7 cells. Western Blot detection of apoptosis marker Cytochrome C was done (top) for EV associated and cellular fractions of HA-HuR and pCIneo (control) expressing Huh 7 cells respectively. Western blot for HA-HuR was done to analyse the expression of HA-HuR in EVs isolated from HA-HuR and pCIneo expressing Huh7 cells. CD63 and β-actin are used as loading control for EV associated fractions and cellular fractions respectively (upper panels). TUNEL assay of HA-HuR and pCIneo expressing and non-transfected Huh 7 cells was performed and representative pictures of HA-HuR and pCIneo expressing cells along with picture of non-transfected but DNase treated (positive control ) cells were compared (lower panels). Scale bar 50 µM.


( B ) Change in miR - 122 level in starved mouse liver . Endogenous miR - 122 ( left panel ) and CAT - 1 mRNA ( middle panel ) levels in mouse liver starved for 12h were quantified by qRT - PCR . miR - 122 levels in the blood serum of corresponding animals were also measured ( right panel ) . The Ct values of miR - 122 and CAT - 1 mRNA obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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(B) Change in miR-122 level in starved mouse liver. Endogenous miR-122 (left panel) and CAT-1 mRNA (middle panel) levels in mouse liver starved for 12h were quantified by qRT-PCR. miR-122 levels in the blood serum of corresponding animals were also measured (right panel). The Ct values of miR-122 and CAT-1 mRNA obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( C ) Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots , β - actin was used as loading control .

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(C)Relative level of HuR in both liver and lung of Fed and Starved mice were analysed by western blots, β-actin was used as loading control .


( E ) Western Blot analysis to check expression of HA - HuR in liver samples of injected mice . β - Actin was used as loading control . HA - HuR expressing Huh7 cells lysate was used as positive control .

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(E) Western Blot analysis to check expression of HA-HuR in liver samples of injected mice. β-Actin was used as loading control. HA-HuR expressing Huh7 cells lysate was used as positive control.


( F ) . Change in miR - 122 level upon expression of HA - HuR in mouse liver . Endogenous miR - 122 level in HA - HuR expression plasmid injected mouse liver were quantified by RT - PCR and compared against liver RNA samples collected from control ( pCIneo injected ) group of animals ( mean + / - s . e . m . , n = 5 ) .

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(F). Change in miR-122 level upon expression of HA-HuR in mouse liver. Endogenous miR-122 level in HA-HuR expression plasmid injected mouse liver were quantified by RT-PCR and compared against liver RNA samples collected from control (pCIneo injected) group of animals (mean+/- s.e.m., n=5).


( G ) Endogenous miR - 122 target CAT - 1 ( left panel ) and Aldolase ( right panel ) mRNA levels in HA - HuR or pCIneo ( control ) injected mouse liver were quantified by RT - PCR ( mean + / - s . e . m . , n = 4 ) .

(G) Endogenous miR-122 target CAT-1 (left panel) and Aldolase (right panel) mRNA levels in HA-HuR or pCIneo (control) injected mouse liver were quantified by RT-PCR (mean+/- s.e.m., n=4).


( H ) miR - 122 levels in the blood serum of pCIneo ( control ) or HA - HuR expression plasmid injected mice were measured . The Ct values of miR - 122 and CAT - 1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted ( mean + / - s . e . m . , n = 5 ) .

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(H) miR-122 levels in the blood serum of pCIneo (control) or HA-HuR expression plasmid injected mice were measured. The Ct values of miR-122 and CAT-1 and Aldolase mRNAs obtained for each individual animal liver and blood serum were plotted (mean+/- s.e.m., n=5).


( A ) Time course experiment of HuR binding of miR - 122 replaced from Ago2miRNPs . A schematic representation of in vitro miRNA binding assay of HuR ( left panel ) . Equal amounts of recombinant HuR ( rHuR ) , after indicated time of the miRNP interaction on FLAG - beads , were immunoprecipitated and HuR associated miR - 122 levels were detected by qRT - PCR ( right top panel ) . Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials . HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials , at different time points ( right bottom panel ) .

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(A) Time course experiment of HuR binding of miR-122 replaced from Ago2miRNPs. A schematic representation of in vitro miRNA binding assay of HuR (left panel). Equal amounts of recombinant HuR (rHuR), after indicated time of the miRNP interaction on FLAG-beads, were immunoprecipitated and HuR associated miR-122 levels were detected by qRT-PCR (right top panel). Ago2western blot detects its presence in the FLAG beads used in the assay and its absence in HuRimmunoprecipitated materials. HuRwestern blot was used to confirm its presence and quantification of its levels in the immunoprecipitated materials, at different time points (right bottom panel).


( C ) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay . The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation . The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti - HuR antibody .

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(C) Coomassie stained gel picture denotes the purity of the recombinant HuR used in the assay. The same proteins were western blotted for HuR to show the specificity of the antibody used for immunoprecipitation. The recombinant P protein and N protein of Chandipura virus was used as negative control to confirm the specificity of 3A2 anti-HuR antibody.


( D ) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials . A representative domain picture of HuR and HuRΔIII are shown in the upper panel . Relative levels of HuR bound miR - 122 were determined by qRT - PCR ( mean + / - s . e . m . , n = 3 ) ( middle left panel ) . Amount of miR - 122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials . Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials ( bottom panel ) .

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(D) HuR western blot in middle right panel confirmed the presence of HuR and the truncated version of it in the immunoprecipitated materials. A representative domain picture of HuR and HuRΔIII are shown in the upper panel. Relative levels of HuR bound miR-122 were determined by qRT-PCR (mean+/- s.e.m., n=3) (middle left panel). Amount of miR-122 recovered from the immunoprecipitated materials were normalized against the amount of HuR or HuRΔIII present in the Immunoprecipitated materials. Ago2 western blot confirmed its presence in the FLAG beads used in the assay while Ago2 was not detected in HuR immunoprecipitated materials (bottom panel).


( E ) RNA electrophoretic mobility shift assay done with 32P - labeled synthetic miR - 122 and recombinant HuR proteins . Radiolabelled miR - 122 ( 100 fmol ) was incubated with increasing concentration of FL - HuR ( 5 - 100nM ) or HuRΔIII ( 100 nM ) and gel shifting of miR - 122 were marked by arrowhead . Position of the free probes is marked by an arrow . In the right panel , 1pmol of unlabelled TNF - alpha AU - rich element encoding synthetic RNA was used to compete with miR - 122 binding of full length HuR . Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page .

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(E) RNA electrophoretic mobility shift assay done with 32P-labeled synthetic miR-122 and recombinant HuR proteins. Radiolabelled miR-122 (100 fmol) was incubated with increasing concentration of FL-HuR (5-100nM) or HuRΔIII (100 nM) and gel shifting of miR-122 were marked by arrowhead. Position of the free probes is marked by an arrow. In the right panel, 1pmol of unlabelled TNF-alpha AU-rich element encoding synthetic RNA was used to compete with miR-122 binding of full length HuR. Amount of HuR or HuRΔIII used for gel shift assay were premeasured by densitometric estimation of coomassie stained gel bands from SDS page.


( F ) Association of miRNAs with recombinant HuR in vitro . Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA , recovered from immunoprecipitated HuR after the binding reaction , were quantified along with the input RNA used for binding . The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted ( mean + / - s . e . m . , n = 3 ) . The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot .

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(F) Association of miRNAs with recombinant HuR in vitro. Small RNA pool of Huh7 cell isolated by miRVana miRNA isolation kit were incubated with recombinant HuR in an in vitro binding reaction and HuR associated RNA, recovered from immunoprecipitated HuR after the binding reaction, were quantified along with the input RNA used for binding. The mean Ct values of RNA from samples from three independent binding reactions along with input RNA were measured and plotted (mean+/- s.e.m., n=3). The Input recombinant HuR used in the binding assay and in the immunoprecipitated materials were detected by western blot.


( A ) Subcellular distribution of HuR and Ago2 in Huh7 cells . Isotonic lysates of Huh7 cells transfected with HA - HuR expression or pCIneo control vector were analyzed on 3 - 30 % OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis . Alix and Calnexin were used as markers of late endosome / MVB and ER respectively . Positions of specific organelle containing fractions were marked above the panels . * denotes the position of the high molecular weight HuR bands detected in the MVB fractions .

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(A) Subcellular distribution of HuR and Ago2 in Huh7 cells. Isotonic lysates of Huh7 cells transfected with HA-HuR expression or pCIneo control vector were analyzed on 3-30% OptiprepTM gradients for separation of organelles and localization of Ago2 and HuR were determined by western blotting analysis. Alix and Calnexin were used as markers of late endosome/MVB and ER respectively. Positions of specific organelle containing fractions were marked above the panels. * denotes the position of the high molecular weight HuR bands detected in the MVB fractions.


( B ) Detection of ubiquitinated HuR in Huh7 cells expressing HA - Ub . HuR was immunoprecipitated with anti - HuR antibody from cell lysate of Huh7 cells transfected with HA - Ub expression or pCIneo ( control ) plasmids and western blotted for HA or HuR ( left panel ) . In vitro ubiquitination assay was done with recombinant His - tagged HuR and cell extract of Huh7 cells expressing HA - Ub . The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown . Specific bands of Ub - HuR are marked by arrows and HuR is marked by arrowhead .

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(B) Detection of ubiquitinated HuR in Huh7 cells expressing HA-Ub. HuR was immunoprecipitated with anti-HuR antibody from cell lysate of Huh7 cells transfected with HA-Ub expression or pCIneo (control) plasmids and western blotted for HA or HuR (left panel). In vitro ubiquitination assay was done with recombinant His-tagged HuR and cell extract of Huh7 cells expressing HA-Ub. The scheme of the experiment and western blot data for input and cell extract along with immunoprecipitated materials are shown. Specific bands of Ub-HuR are marked by arrows and HuR is marked by arrowhead.


( C ) Changes in relative level of miR - 122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA - HuR expression vector transfected cells were measured by qRT - PCR ( mean + / - s . e . m . , n = 5 ) .

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(C) Changes in relative level of miR-122 in the subcellular ER and MVB associated fractions of OptiprepTM gradients done with the cell lysates from pCIneo or HA-HuR expression vector transfected cells were measured by qRT-PCR (mean+/- s.e.m., n=5).


( D ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells . Organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR . Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins . Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads . Levels of HuR in the immunoprecipitated materials were detected using anti - HuR antibody . Anti - GFP antibody immnoprecipitated materials were used as control .

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(D) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of Huh7 cells. Organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for ubiquitin to detect ubiquitinated form of HuR. Whole cell lysates used for optiprep analysis were western blotted for ubiquitinated proteins. Positions of the high molecular weight bands of ubiquitinated HuR are marked by arrowheads. Levels of HuR in the immunoprecipitated materials were detected using anti-HuR antibody. Anti-GFP antibody immnoprecipitated materials were used as control.


( E ) Detection of ubiquitinated HuR in HuR - immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co - expressing HA - Ubiquitin and FLAG - HuR . Cells were lysed and organelles were separated on OptiprepTM gradients , and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR . HuR in the lysate of the FLAG - HuR and HA - Ub co expressing cells were detected by HuR specific antibody .

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(E) Detection of ubiquitinated HuR in HuR-immunoprecipitated materials obtained from ER and MVB enriched fractions of cells co-expressing HA-Ubiquitin and FLAG-HuR. Cells were lysed and organelles were separated on OptiprepTM gradients, and HuR in organeller lysates were immunoprecipitated with HuR specific antibody and western blotted for HA to detect ubiquitinated form of HuR. HuR in the lysate of the FLAG-HuR and HA-Ub co expressing cells were detected by HuR specific antibody.


( F ) Loss of miRNA association of HuR on MVB . HA - HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA - HuR and associated miRNA levels were measured by qRT - PCR and normalized against immunoprecipitated proteins . Levels of immunoprecipitated HA - HuR were detected by western blotting and quantified by densitometry . Data represented as mean + / - s . e . m . , n = 3 .

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(F) Loss of miRNA association of HuR on MVB. HA-HuR expressing Huh7 cell lysates separated on OptiprepTM gradients were used for immunoprecipitation of HA-HuR and associated miRNA levels were measured by qRT-PCR and normalized against immunoprecipitated proteins. Levels of immunoprecipitated HA-HuR were detected by western blotting and quantified by densitometry. Data represented as mean+/- s.e.m., n=3.


( G ) Loss of miRNA from ubiquitinated form of HuR . Scheme of the double immunoprecipitation experiment is shown in the left panel . Western blot analysis for HuR in the anti - HA and anti - HuRimmunoprecipitatedFLAG - HuR along with the input of FLAG - HuR used . Lysates of cell expressing the FLAG - HuR and used for FLAGHuR purification is also shown ( right upper panels ) . qRT - PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots ( right lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . Position of ubiquitinated band of FLAG - HuR is marked by arrow .

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

(G) Loss of miRNA from ubiquitinated form of HuR. Scheme of the double immunoprecipitation experiment is shown in the left panel. Western blot analysis for HuR in the anti-HA and anti-HuRimmunoprecipitatedFLAG-HuR along with the input of FLAG-HuR used. Lysates of cell expressing the FLAG-HuR and used for FLAGHuR purification is also shown (right upper panels). qRT-PCR based estimation of the miRNA per unit of HuRimmunoprecipitated were estimated for both HA and HuRimmunoprecipitated materials and normalized against respective protein levels quantified by densitometric quantification of western blots (right lower panel). Data represented as mean+/- s.e.m., n=3. Position of ubiquitinated band of FLAG-HuR is marked by arrow.


( H ) Increase in ubiquitinated form of HuR in Starved Huh7 cells . Levels of ubiquitinated HuR in Fed and Starved ( 5h ) HA - Ub expressing Huh7 cells treated either with ethanol or MG132 ( 20µM ) for same duration . The lysates were immunoprecipitated with HuR and western blotted for HA and HuR . Position of ubiquitinated bands were marked by arrows . The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non - ubiquitinated form were measured and plotted ( mean + / - s . e . m . , n = 3 ) .

O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Increase in ubiquitinated form of HuR in Starved Huh7 cells. Levels of ubiquitinated HuR in Fed and Starved (5h) HA-Ub expressing Huh7 cells treated either with ethanol or MG132 (20µM) for same duration. The lysates were immunoprecipitated with HuR and western blotted for HA and HuR. Position of ubiquitinated bands were marked by arrows. The levels of ubiquitinated HuR were measured by densitometry and relative increase in ubiquitinated HuR levels against the non-ubiquitinated form were measured and plotted (mean+/- s.e.m., n=3).


( I ) Full length and a truncated version of HA - HuR without the hinge region ( graphical scheme in upper panel ) was used to score the effect of deletion of hinge region on EV associated miR - 122 content measured by qRT - PCR ( bottom panel ) . Western blots to check expression of HuR and its truncated variants are also shown . " * " denotes the HA - HuR band ( left lower panel ) . Data represented as mean + / - s . e . m . , n = 3 .

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

(I) Full length and a truncated version of HA-HuR without the hinge region (graphical scheme in upper panel) was used to score the effect of deletion of hinge region on EV associated miR-122 content measured by qRT-PCR (bottom panel). Western blots to check expression of HuR and its truncated variants are also shown." * " denotes the HA-HuR band (left lower panel). Data represented as mean+/- s.e.m., n=3.


( J ) Ubiquitination status of full length and a truncated version of HA - HuR without the hinge region between RRMII and RRMIII in Huh7 cells . HuR ( full lenght or truncated version ) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA - HuR and ubiquitination . Ubiquinitated HuR bands are marked by arrows . postion of non - ubiquitinated proteins were marked by arrowheads ( upper panel ) . qRT - PCR based estimation of associated miRNA per unit of HA - HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots ( lower panel ) . Data represented as mean + / - s . e . m . , n = 3 . PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR - 122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells .

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

(J) Ubiquitination status of full length and a truncated version of HA-HuR without the hinge region between RRMII and RRMIII in Huh7 cells. HuR (full lenght or truncated version) were immunoprecipitated from cell lysates of Huh7 cells expressing these proteins and the immunoprecipitated materials were western blotted for HA-HuR and ubiquitination. Ubiquinitated HuR bands are marked by arrows. postion of non-ubiquitinated proteins were marked by arrowheads (upper panel). qRT-PCR based estimation of associated miRNA per unit of HA-HuR immunoprecipitated materials were estimated and normalized against respective protein levels quantified by densitometric quantification of western blots (lower panel). Data represented as mean+/- s.e.m., n=3. PCIneo transfected cells were used as control in immunoprecipitation reaction but no amplification of miR-122 were detected for HA immunoprecipitated materials from pCIneo tranfected cells.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with ( A ) a murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with (A) a murine macrophage cell line (J774.2) treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with a human macrophage cell line ( U937 ) , treated either with thapsigargin ( black bars ) , tunicamycin ( red bars ) or DMSO ( white bars ) . Graphs show mean ± SEM from n = 3biological replicates . * * * * P < 0 . 0001 ( Unpaired 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 B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with a human macrophage cell line (U937), treated either with thapsigargin (black bars),tunicamycin (red bars) or DMSO (white bars). Graphs show mean ± SEM from n=3biological replicates. ****P < 0.0001 (Unpaired t test).


Heat map represents cytokine secretion by in vitro expanded ( C ) murine splenic iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded (C) murine splenic iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co - cultured either with thapsigargin - or tunicamycin - or DMSO - treated murine BMDMs . The heat map shows the average result of two pooled biological replicates .

O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

Heat map represents cytokine secretion by in vitro expanded human peripheral blood iNKT cells co-cultured either with thapsigargin- or tunicamycin- or DMSO-treated murine BMDMs. The heat map shows the average result of two pooled biological replicates.


Wild type C57BL / 6 mice were injected intravenously with thapsigargin - loaded PLGA nanoparticles or vehicle , and 12 h later , hepatic iNKT cells were analysed by flow cytometry . Histograms represent expression of intracellular IL - 4 and IFN - γ levels in hepatic iNKT cells from mice injected with thapsigargin - loaded PLGA nanoparticles ( red histogram ) or vehicle - treated ( grey histogram ) mice . Dot plots represents MFI for intracellular IL - 4 and IFN - γ expression in hepatic iNKT cells from thapsigargin - loaded PLGA nanoparticles ( red dots ) or vehicle - treated ( black dots ) mice . Data plots show mean ± SEM from n = 5 mice per group . * * P ≤ 0 . 001 ( Mann - Whitney U test ) .

O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O 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-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type C57BL/6 mice were injected intravenously with thapsigargin-loaded PLGA nanoparticles or vehicle, and 12 h later, hepatic iNKT cells were analysed by flow cytometry. Histograms represent expression of intracellular IL-4 and IFN-γ levels in hepatic iNKT cells from mice injected with thapsigargin-loaded PLGA nanoparticles (red histogram) or vehicle-treated (grey histogram) mice. Dot plots represents MFI for intracellular IL-4 and IFN-γ expression in hepatic iNKT cells from thapsigargin-loaded PLGA nanoparticles (red dots) or vehicle-treated (black dots) mice. Data plots show mean ± SEM from n = 5 mice per group. **P≤ 0.001 (Mann-Whitney U test).


Thapsigargin or αGalCer treated J774 . 2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d - specific antibody ( black circles ) or isotype ( red squares ) for 16 hours . The addition of an anti - CD1d antibody blocked the activation of 2C12 demonstrating that the NKT - cell activation was CD1d - specific . Graphs show mean ± SEM from n = 2 biological replicates .

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

Thapsigargin or αGalCer treated J774.2 cells were cocultured with 2C12 in the presence or absence of the murine CD1d-specific antibody (black circles) or isotype (red squares) for 16 hours. The addition of an anti-CD1d antibody blocked the activation of 2C12 demonstrating that the NKT-cell activation was CD1d-specific. Graphs show mean ± SEM from n=2 biological replicates.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin - or tunicamycin - treated CD1d knockdown ( white bars ) or untransfected ( black bars ) . Knockdown of CD1d blocked IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured murine macrophage cell line (J774.2) treated either with thapsigargin- or tunicamycin-treated CD1d knockdown (white bars) or untransfected (black bars). Knockdown of CD1d blocked IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Bar graph represents IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured either with thapsigargin - or tunicamycin - treated primary BMDM isolated from CD1d - / - ( white bars ) or wild type ( black bars ) mice . 2C12 cocultured with CD1d - / - BMDMs resulted in reduced IL - 2 secretion . Graphs show mean ± SEM from n = 3 biological replicates . * * * P < 0 . 001 ( Unpaired t test ) .

Bar graph represents IL-2 secretion by NKT cell hybridoma (2C12) co-cultured either with thapsigargin- or tunicamycin-treated primary BMDM isolated from CD1d-/- (white bars) or wild type (black bars) mice. 2C12 cocultured with CD1d-/- BMDMs resulted in reduced IL-2 secretion. Graphs show mean ± SEM from n=3 biological replicates. ***P < 0.001 (Unpaired t test).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


qPCR analyses showing the expression ( fold induction ) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin , tunicamycin or DMSO for 4 or 8 hours . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of UPR signature genes compared to DMSO in human macrophage cell lines U937 stimulated either with thapsigargin, tunicamycin or DMSO for 4 or 8 hours. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with murine macrophage cell line ( J774 . 2 ) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor ( 4μ8c ) ( black bar ) or PERK inhibitor ( GSK ) ( grey bar ) or ATF6α inhibitor ( Ceapin - a7 ) ( red bar ) respectively . Inhibition of IRE1α or PERK resulted in significant reduction in IL - 2 secretion by iNKT hybridoma compared to controls . In total , n = 2 biological replicates were performed ( two and four technical replicates per biological replicate respectively ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with murine macrophage cell line (J774.2) treated either with thapsigargin or tunicamycin in the presence of IRE1α inhibitor (4μ8c) (black bar) or PERK inhibitor (GSK) (grey bar) or ATF6α inhibitor (Ceapin-a7) (red bar) respectively. Inhibition of IRE1α or PERK resulted in significant reduction in IL-2 secretion by iNKT hybridoma compared to controls. In total, n=2 biological replicates were performed (two and four technical replicates per biological replicate respectively). Graphs show mean ± SEM of each biological replicate.


Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774 . 2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene .

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

Immunoblots represents the knockdown efficiency for IRE1α and PERK protein in J774.2 cell line transduced with lentiviral particles carrying gRNA against the IRE1α or PERK gene.


Bar graph represents percentage of IL - 2 secretion by NKT cell hybridoma ( 2C12 ) co - cultured with thapsigargin - treated murine IRE1α or PERK knockdown macrophage cell line ( J774 . 2 ) . Knockdown of IRE1α ( black bar ) or PERK ( grey bar ) resulted in significant reduction in IL - 2 secretion by iNKT hybridoma . In total n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents percentage of IL-2 secretion by NKT cell hybridoma (2C12) co-cultured with thapsigargin-treated murine IRE1α or PERK knockdown macrophage cell line (J774.2). Knockdown of IRE1α (black bar) or PERK (grey bar) resulted in significant reduction in IL-2 secretion by iNKT hybridoma. In total n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 6 ) were isolated from J774 . 2 macrophages stimulated with thapsigargin , tunicamycin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or tunicamycin - treated ( red bars ) J774 . 2 cells . Polar lipid fractions induce very little IL - 2 secretion by 2C12 . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-6) were isolated from J774.2 macrophages stimulated with thapsigargin, tunicamycin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents NKT cell activation by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or tunicamycin-treated (red bars) J774.2 cells. Polar lipid fractions induce very little IL-2 secretion by 2C12. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin - treated J774 . 2 at acidic pH and reduced mCD1d loading at neutral pH . Graphs show mean ± SEM from n = 2 biological replicates ( two technical replicates per biological replicate ) .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Graph represents enhancement of loading with neutral lipid fractions isolated from thapsigargin-treated J774.2 at acidic pH and reduced mCD1d loading at neutral pH. Graphs show mean ± SEM from n=2 biological replicates (two technical replicates per biological replicate).


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) , isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges , were co - cultured with purified NKT1 subsets . Bar graphs represent IFN - γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) primary murine macrophages . Polar lipid fractions induce a minimal amount of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5), isolated from murine bone marrow derived macrophages stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges, were co-cultured with purified NKT1 subsets. Bar graphs represent IFN-γ release by NKT1 cells induced by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) primary murine macrophages. Polar lipid fractions induce a minimal amount of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Neutral ( Fractions 1 - 3 ) and polar lipids ( Fractions 4 - 5 ) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid - phase extraction ( SPE ) cartridges . Bar graph represents IFN - γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin - ( black bars ) or DMSO - treated ( white bars ) human primary macrophages . Polar lipid fractions induce only minimal amounts of IFN - γ secretion by the NKT1 sublineage . In total , n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs shows mean ± SEM of each biological replicate .

O O O O 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-CONTROLLED_VAR O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Neutral (Fractions 1-3) and polar lipids (Fractions 4-5) were isolated from primary macrophages derived from human peripheral blood stimulated with thapsigargin or DMSO and fractionated using solid-phase extraction (SPE) cartridges. Bar graph represents IFN-γ release by NKT1 cells by CD1d protein bound neutral lipids fractions isolated from thapsigargin- (black bars) or DMSO-treated (white bars) human primary macrophages. Polar lipid fractions induce only minimal amounts of IFN-γ secretion by the NKT1 sublineage. In total, n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs shows mean ± SEM of each biological replicate.


Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL - 2 secretion by NKT cells . In total , n = 2 biological replicates were performed ( three technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

Bar graph represents that recycling of CD1d between various endomembrane compartments and the cell surface is required for optimal IL-2 secretion by NKT cells. In total, n=2 biological replicates were performed (three technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


qPCR analyses showing the expression ( fold induction ) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774 . 2 stimulated either with thapsigargin or DMSO for 8 hours . In total n = 2 biological replicates were performed ( two technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qPCR analyses showing the expression (fold induction) of genes encoding for glycosphingolipid degrading enzymes compared to DMSO in murine macrophage cell lines J774.2 stimulated either with thapsigargin or DMSO for 8 hours. In total n=2 biological replicates were performed (two technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells . IL - 2 secretion in UPR - induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin . Graphs show mean ± SEM from n = 3 biological replicates . n . s ( non - significant ) ( One - way ANOVA ) biological replicates .

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

Glycolipid processing in intracellular compartments is not required prior to presentation to NKT cells. IL-2 secretion in UPR-induced NKT cell activation in the presence or absence of castanospermine or deoxygalactonojirimycin. Graphs show mean ± SEM from n = 3 biological replicates. n.s (non-significant) (One-way ANOVA) biological replicates.


L363 antibody does not impact UPR - induced NKT cell activation . 2C12 NKT cells were co - cultured with ER - stressed macrophages J774 . 2 cells and treated with L363 antibody or isotype control . α - GalCer induced responses served as positive control . In total , n = 2 biological replicates were performed ( Four technical replicates per biological replicate ) . Graphs show mean ± SEM of each biological replicate .

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

L363 antibody does not impact UPR-induced NKT cell activation. 2C12 NKT cells were co-cultured with ER-stressed macrophages J774.2 cells and treated with L363 antibody or isotype control. α-GalCer induced responses served as positive control. In total, n=2 biological replicates were performed (Four technical replicates per biological replicate). Graphs show mean ± SEM of each biological replicate.


Representative confocal 3D images of LysoTracker ( red ) , DAPI ( blue ) , and BODIPY ( green ) stained ATM subsets obtained from eWAT of HFD ( 16 weeks ) treated mice ( n = 8 mice from two independent experiments ) . The merged figures are the result of the overlap of all three fluorochromes . Scale bar , 10 μm .

Representative confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.


Mito Stress assay of sorted ATM subsets ( red , MHCIIlow ; green , MHCIIhi ; and blue , CD11c + ) from 16 - week - HFD obese mice showing overall oxygen consumption rate ( OCR ) , maximal respiration , spare capacity respiration , basal respiration , ATP production , and non - mitochondrial respiration . OCR data shown are representative of four independent experiments and expressed as mean ± SD ( n = 4 MHCIIlow ; n = 7 MHCIIhi ; n = 5 CD11c + ) , with statistical significance determined using one - way ANOVA test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : no significant difference ) .

O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).


Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169 - DTR ND - ( A ) or HFD - treated ( 16 weeks ) ( B ) mice . A representative analysis of three independent experiments is 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

Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown.


eWAT was collected from lean ( C ) and obese ( HFD 16 weeks ) ( D ) WT or CD169 - DTR mice after 12 days DT - mediated ATM depletion . Representative pictures of dissected eWAT are shown on the left , the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right , with statistical significance calculated using the Student ' s t - test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . The zoomed - in area shows clear , oily lipid accumulation in the absence of ATMs ( lower left part ) .

O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O

eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).


Adipocyte hypertrophy is visible in the absence of ATMs . Representative images of hematoxylin and eosin staining ( H & E ) ( E ) and corresponding adipocyte diameters ( F ) of lean and obese ( HFD 16 weeks ) eWAT from WT or CD169 - DTR mice treated for 7 or 12 days with DT . Scale bar , 50 μm . Each group comprised 3 - 5 mice . Statistical significance was determined using one - way ANOVA test . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; ns : no significant difference . The box and whisker plots show the median value and 10 - 90 percentiles of adipocyte diameters .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.


qPCR showing the relative expression of genes for adipogenesis ( Pparγ ) , lipolysis ( Lpl ) , and lipogenesis ( Dgat1 ) , plus other lipid receptor / transporters ( Cd36 , Abca1 ) or chaperones ( Fabp4 ) in obese ( HFD 16 weeks ) eWAT of CD169 - DTR ( red bars ) mice and DT - injected WT controls ( blue bars ) ( upper panel ) and obese eWAT of anti - CSF1R antibody - injected mice ( green bars ) mice and isotype control - injected WT controls ( white bars ) ( lower panel ) . Mice were under DT or anti - CSF1R antibody treatment for 12 days . ( n = 5 pooled mice per group , mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significant difference .

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

qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.


Violin plots depicting the expression of representative marker genes for each CD45 - cell cluster : ASC1 ( Gsn , Pdgfrb , Eln ) ( red ) , ASC2 ( Pi16 , Pdgfra , Cebpd ) ( yellow ) , stromal cells ( Col1a1 , Col1a2 , Myl9 ) ( green ) , VEC1 / VEC2 ( Pecam - 1 , Cldn5 , Aqp1 ) ( purple and blue ) , and mesothelial - like cells ( MLCs ) ( Upk3b , Msln , Acta2 ) ( orange ) .

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

Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange).


Representative 3D fluorescence imaging of lean and obese ( HFD 16 weeks ) eWAT samples stained with anti - MHCII ( green in lean samples ) , anti - CD11c ( green in obese samples ) , anti - CD31 ( blue ) , anti - CD169 ( pink ) , and DAPI ( white ) . The two images at the bottom show the images merged for the lean and obese conditions without DAPI . Scale bar : 100 μm ( lean ) and 70 μm ( obese ) .

Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese).


Higher magnification of specific eWAT areas . Scale bar : 30 μm ( lean ) and 50 μm ( obese ) . ( left panels ) Distance of different ATM subsets to blood vessels . Distances ( in μm ) were calculated using the Imaris software , as described in the Materials and Methods section . The box and whisker plots show the median value and 10 - 90 percentiles of distance values . The right panels show the distribution of ATM subsets to blood vessels within a certain distance ( ≤ 1 μm , 1 - 10 μm , ≥ 10 μm ) . C : CD169 + MHCIIlow and CD169 + MHChi ATMs in lean eWAT . D : CD169 + CD11c - and CD169 + CD11c + ATMs in obese ( HFD 16 weeks ) eWAT . Four mice from two independent experiments were analyzed ( n = 4 - 5 ; mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * * * P < 0 . 0001 , bars with no asterisks showed no significant difference .

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

Higher magnification of specific eWAT areas. Scale bar: 30 μm (lean) and 50 μm (obese). (left panels) Distance of different ATM subsets to blood vessels. Distances (in μm) were calculated using the Imaris software, as described in the Materials and Methods section. The box and whisker plots show the median value and 10-90 percentiles of distance values. The right panels show the distribution of ATM subsets to blood vessels within a certain distance (≤1 μm, 1-10 μm, ≥10 μm). C: CD169+MHCIIlow and CD169+MHChi ATMs in lean eWAT. D: CD169+CD11c- and CD169+CD11c+ ATMs in obese (HFD 16 weeks) eWAT. Four mice from two independent experiments were analyzed (n = 4-5; mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, ****P < 0.0001, bars with no asterisks showed no significant difference.


Model construction based on 3D reconstructed images showing the relative distance of CD11c + ATMs from traced large blood vessels . Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing ( white filaments ) . CD11c + ATMs are close ( < 10µm ) to the traced large blood vessels ( red ellipsoids ) or far ( > 10 µm ) ( grey ellipsoids ) from the traced blood vessel . Blood vessels are shown in green . Scale bar : 100 μm . The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤ 10 or > 10 μm to blood vessels quantified from 3 biological replicates from 1 experiment , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 .

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

Model construction based on 3D reconstructed images showing the relative distance of CD11c+ ATMs from traced large blood vessels. Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing (white filaments). CD11c+ ATMs are close (< 10µm) to the traced large blood vessels (red ellipsoids) or far (>10 µm) (grey ellipsoids) from the traced blood vessel. Blood vessels are shown in green. Scale bar: 100 μm. The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤10 or >10 μm to blood vessels quantified from 3 biological replicates from 1 experiment, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Two representative images of eWAT dissected from Evans blue - injected obese ( HFD 16 weeks ) WT and CD169 - DTR mice treated for 7 days with DT . Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm ( the absorption values were normalized to the tissue weight in grams ) . A chart showing the normalized mean optical density ± SD ( n = 5 - 7 mice per group ) . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 .

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

Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Mmp12 ( H ) and Vegfα ( F ) are highly expressed in MHCIIhi and CD11c + ATMs purified from obese ( HFD 16 weeks ) eWAT . qPCR analysis showed the relative expression in all three ATM subsets ( n = 3 ) , data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one - way ANOVA test . * P < 0 . 05 , * * P < 0 . 01 . qPCR analysis showing the relative expression of Vegfα in obese ( HFD 16 weeks ) eWAT of WT ( n = 3 ) or CD169 - DTR mice ( n = 5 ) treated for 7 days with DT , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * * P < 0 . 0001 .

Mmp12 (H) and Vegfα (F) are highly expressed in MHCIIhi and CD11c+ ATMs purified from obese (HFD 16 weeks) eWAT. qPCR analysis showed the relative expression in all three ATM subsets (n=3), data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one-way ANOVA test. *P < 0.05, **P < 0.01. qPCR analysis showing the relative expression of Vegfα in obese (HFD 16 weeks) eWAT of WT (n=3) or CD169-DTR mice (n=5) treated for 7 days with DT, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001.


Elastin staining showing more pronounced ECM accumulation in eWAT of CD169 - DTR ( HFD 16 weeks ) when compared with ECM accumulation in the WT obese ( HFD 16 weeks ) control ( DAPI , blue ; elastin , red ; CD31 , green ) . The fourth image in each row shows the three channels merged . Scale bar : 50 μm . Picrosirius red - staining depicts excessive collagen deposits in obese ( HFD 16 weeks ) eWAT of CD169 - DTR mice when compared with collagen deposits in WT controls . Scale bar : 250 μm . Upper panels : brightfield microscopy ; lower panels : fluorescence microscopy .

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Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.


Violin plots showing the pro - inflammatory cytokine profile ( Il - 6 , Tnf - α , and Il - 1β ) of all 23 cell clusters obtained from obese ( HFD 16 weeks ) WT and CD169 - DTR eWAT . Cluster 0 : ASC1 ; Cluster 1 : Stromal cells ; Cluster 2 : VEC1 ; Cluster 3 : VEC2 ; Cluster 4 : NK1 ; Cluster 5 : MAC / Mono1 ; Cluster 6 : ASC2 ; Cluster 7 : B ; Cluster 8 : cDC1 ; Cluster 9 : mono - derived cDC ; Cluster 10 : Th2 / ILC2 / Treg ; Cluster 11 : MAC / Mono3 ; Cluster 12 : Th17 ; Cluster 13 : VEC2 ; Cluster 14 : CD8 ; Cluster 15 : MLCs ; Cluster 16 : CD11b + DC ; Cluster 17 : Neutrophils ; Cluster 18 : cDC2 ; Cluster 19 : cDC ; Cluster 20 : NKT ; Cluster 21 : NK2 ; Cluster 22 : Mast cells / Basophils . ASC : adipocyte stem cell ; VEC : vascular endothelial cell ; MLC : mesothelial - like cell ; NK : natural killer ; MAC : macrophage ; Mono : monocyte ; DC : dendritic cell ; NKT : natural killer T cells .

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

Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells.


( A ) Confocal laser scanning micrographs showing the localization of a functional CDKA ; 1 : mVenus fusion protein in the wildtype ( WT ) and cartoons on top highlighting the changes in abundance of CDKA ; 1 : mVenus in the nucleus and cytoplasm during the course of meiosis . The region colored in beige represents the cytoplasm , in green the nucleoplasm , and in white the nucleolus . Scale bar : 10 μm .

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(A) Confocal laser scanning micrographs showing the localization of a functional CDKA;1:mVenus fusion protein in the wildtype (WT) and cartoons on top highlighting the changes in abundance of CDKA;1:mVenus in the nucleus and cytoplasm during the course of meiosis. The region colored in beige represents the cytoplasm, in green the nucleoplasm, and in white the nucleolus. Scale bar: 10 μm.


( B ) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA ; 1 : mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis . Error bars represent mean ± SD and two biological replicates were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative analysis of the signal distribution of the nuclear versus cytoplasmic fraction of CDKA;1:mVenus during prophase I of meiosis as revealed by live cell imaging Twenty cells at each time point were used for the analysis. Error bars represent mean ± SD and two biological replicates were performed.


( C ) Immunolocalization of CDKA ; 1 ( green ) and ASY1 ( red ) on spread chromsomes in leptotene and zygotene of wild - type plants expressing a functional PROCDKA ; 1 : CDKA ; 1 : Strep construct . The last lane shows a magnification of the region marked by the red rectangle . Arrowheads indicate synapsed regions of homologous chromosomes where CDKA ; 1 is no longer present . Scale bar : 5 μm .

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(C) Immunolocalization of CDKA;1 (green) and ASY1 (red) on spread chromsomes in leptotene and zygotene of wild-type plants expressing a functional PROCDKA;1:CDKA;1:Strep construct. The last lane shows a magnification of the region marked by the red rectangle. Arrowheads indicate synapsed regions of homologous chromosomes where CDKA;1 is no longer present. Scale bar: 5 μm.


( D ) Chromosome spread analysis of the wildtype and the hypomorphic cdka ; 1 mutant CDKA ; 1T161D . ( a , h ) zygotene or zygotene - like stages ; ( b , i ) pachytene or pachytene - like stages ; ( c , j , k ) diakinesis or dikinesis - like stages ; ( d ) metaphase I ; ( e , i , m , n ) end of meiosis I with two ( e , m ) or three ( i ) pools of chromosomes ; ( f ) metaphase II ; ( g ) tetrad . Red arrowheads indicate the initiated formation of a phragmoplast . White arrowheads depict mitochondria . Scale bars : 10 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

(D) Chromosome spread analysis of the wildtype and the hypomorphic cdka;1 mutant CDKA;1T161D. (a, h) zygotene or zygotene-like stages; (b, i) pachytene or pachytene-like stages; (c, j, k) diakinesis or dikinesis-like stages; (d) metaphase I; (e, i, m, n) end of meiosis I with two (e, m) or three (i) pools of chromosomes; (f) metaphase II; (g) tetrad. Red arrowheads indicate the initiated formation of a phragmoplast. White arrowheads depict mitochondria. Scale bars: 10 μm.


( A ) ASY1 : GFP and ASY1T142D : GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA ; 1T161D mutants . ASY3 : RFP , highlighting chromsomes , was used as a marker for the staging of meiosis . Scale bar : 5 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

(A) ASY1:GFP and ASY1T142D:GFP localization in late G2 and leptotene of male meiocytes of the wildtype and CDKA;1T161D mutants. ASY3:RFP, highlighting chromsomes, was used as a marker for the staging of meiosis. Scale bar: 5 μm.


( B ) Kinase assays of CDKA ; 1 - SDS , - TAM , and - CYCA3 ; 1 complexes using ASY1 puried from baculovirus - infected insect cells as a substrate . The upper panel shows the autoradiograph . The control reaction without CDKA ; 1 - cyclin complex indicates a background activity co - purified from insect cells . The lower panel indicates protein loading by coomassie briliant blue ( CBB ) staining . Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay . Numbers indicate the relative intensities of ASY1 bands .

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(B) Kinase assays of CDKA;1-SDS, -TAM, and -CYCA3;1 complexes using ASY1 puried from baculovirus-infected insect cells as a substrate. The upper panel shows the autoradiograph. The control reaction without CDKA;1-cyclin complex indicates a background activity co-purified from insect cells. The lower panel indicates protein loading by coomassie briliant blue (CBB) staining. Arrowheads indicate ASY1 proteins and asterisks depict the relevant cyclin used which also gets phosphorylated in the assay. Numbers indicate the relative intensities of ASY1 bands.


( C ) The upper panel shows a phos - tag gel analysis of ASY11 - 300 and ASY11 - 300 / T142V ; T184V with and without CDKA ; 1 - SDS kinase complexes using an anti - MBP antibody . The lower panel denotes loading of CDKA ; 1 using an anti - Strep antibody . Arrowheads represent the proteins as indicated .

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(C) The upper panel shows a phos-tag gel analysis of ASY11-300 and ASY11-300/T142V;T184V with and without CDKA;1-SDS kinase complexes using an anti-MBP antibody. The lower panel denotes loading of CDKA;1 using an anti-Strep antibody. Arrowheads represent the proteins as indicated.


( B ) Localization patterns of different ASY1 : GFP variants together with ASY3 : RFP ( for staging of zygotene and pachytene ) in a asy1 mutant background during prophase I . Bar : 5

O O O O O O O O O O O O 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) Localization patterns of different ASY1:GFP variants together with ASY3:RFP (for staging of zygotene and pachytene) in a asy1 mutant background during prophase I. Bar: 5 μ


( C ) Signal distribution profiles of ASY1 : GFP and ASY1T142V : GFP at leptotene The many small peaks with low amplitude in ASY1T142V : GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype .

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(C) Signal distribution profiles of ASY1:GFP and ASY1T142V:GFP at leptotene The many small peaks with low amplitude in ASY1T142V:GFP indicate diffused localization as opposed to the clear peaks seen in the wildtype.


( D , E ) Immunolocalization of ASY1 ( D ) and ZYP1 ( E ) in ASY1 : GFP ( asy1 ) and ASY1T142V : GFP ( asy1 ) plants using anti - GFP and anti - ZYP1 antibodies , respectively . DNA was stained with DAPI ( blue ) . Scale bars : 5 μm .

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(D, E) Immunolocalization of ASY1 (D) and ZYP1 (E) in ASY1:GFP (asy1) and ASY1T142V:GFP (asy1) plants using anti-GFP and anti-ZYP1 antibodies, respectively. DNA was stained with DAPI (blue). Scale bars: 5 μm.


( A ) Yeast two - hybrid interaction assays of ASY3 with different ASY1 variants . Monomeric GFP ( mGFP ) fused with AD ( activating domain ) and BD ( binding domain ) were used as controls . Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp ( - L / T , left panel ) , on Synthetic Drop - out ( SD ) medium in the absence of Leu , Trp , and His ( - L / T / H , middle panel ) , and on SD medium in the absence of Leu , Trp , His and Ade ( - L / T / H / A , right panel ) . Yeast cells were incubated until OD600 = 1 and then diluted 10 - , 100 - and 1000 - fold for the assays .

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(A) Yeast two-hybrid interaction assays of ASY3 with different ASY1 variants. Monomeric GFP (mGFP) fused with AD (activating domain) and BD (binding domain) were used as controls. Yeast cells harboring both the AD and BD plasmids were grown on synthetic medium supplied with glucose in the absence of Leu and Trp (-L/T, left panel), on Synthetic Drop-out (SD) medium in the absence of Leu, Trp, and His (-L/T/H, middle panel), and on SD medium in the absence of Leu,Trp, His and Ade (-L/T/H/A, right panel). Yeast cells were incubated until OD600 = 1 and then diluted 10-, 100- and 1000-fold for the assays.


( B ) GST pull down of ASY3 with different ASY1 variants . The numbers above the bands show the relative intensity of the bands . The input and pull down fractions were analyzed by immuno - blotting with the anti - GST ( ASY3 ) and anti - MBP ( ASY1 ) antibodies . ( C ) Quantification of the pull down fractions of ASY1 as shown in ( B ) . The band intensity in the pull down of ASY11 - 300 / T142V ; T184V at a Triton X - 100 concentration of 0 . 5 % was defined as 1 . The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled - down ASY3 fraction . The average band intensity of ASY1 at different concentrations of Triton X - 100 used was plotted . Asteriks indicate significant difference ( two - tailed t - test , p < 0 . 05 ) . Error bars represent mean ± SD and two biological replicates were performed . .

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(B) GST pull down of ASY3 with different ASY1 variants. The numbers above the bands show the relative intensity of the bands. The input and pull down fractions were analyzed by immuno-blotting with the anti-GST (ASY3) and anti-MBP (ASY1) antibodies. (C) Quantification of the pull down fractions of ASY1 as shown in (B). The band intensity in the pull down of ASY11-300/T142V;T184V at a Triton X-100 concentration of 0.5% was defined as 1. The relative amount of ASY1 in the pull down fractions was normalized by the band intensity of the pulled-down ASY3 fraction. The average band intensity of ASY1 at different concentrations of Triton X-100 used was plotted. Asteriks indicate significant difference (two-tailed t-test, p < 0.05). Error bars represent mean ± SD and two biological replicates were performed..


( A ) Localization patterns of ASY1T142V ; T184V : GFP together with ASY3 - RFP in the wildtype and pch2 mutants . Please note that images of ASY1T142V ; T184V : GFP in pch2 mutants were taken with increased sensitivity for a better visibility . Scale bar : 5 μm .

(A) Localization patterns of ASY1T142V;T184V:GFP together with ASY3-RFP in the wildtype and pch2 mutants. Please note that images of ASY1T142V;T184V:GFP in pch2 mutants were taken with increased sensitivity for a better visibility. Scale bar: 5 μm.


( B ) Seed sets ( mean ± SD , n = 5 ) of WT , asy1 , pch2 , asy1 pch2 , ASY1T142V ; T184V : GFP ( asy1 ) and ASY1T142V ; T184V : GFP ( asy1 pch2 ) plants . Asteriks indicate significant differences ( two - tailed t - test , P < 0 . 01 ) and ns depicts no significant difference . Scale bar : 2 mm .

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(B) Seed sets (mean ± SD, n = 5) of WT, asy1, pch2, asy1 pch2, ASY1T142V;T184V:GFP (asy1) and ASY1T142V;T184V:GFP (asy1 pch2) plants. Asteriks indicate significant differences (two-tailed t-test, P < 0.01) and ns depicts no significant difference. Scale bar: 2 mm.


( C ) Localization patterns of ASY1 : GFP , ASY1T142V ; T184V : GFP , and ASY11 - 570 : GFP in the wildtype ( WT ) and / or in pch2 mutant plants at early prophase I . Scale bars : 5 μm .

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(C) Localization patterns of ASY1:GFP, ASY1T142V;T184V:GFP, and ASY11-570:GFP in the wildtype (WT) and/or in pch2 mutant plants at early prophase I. Scale bars: 5 μm.


( D ) Localization pattern of PCH2 : GFP together with ASY1 : RFP in the male meiocytes of wildtype . Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2 . Scale bar : 10 μm .

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(D) Localization pattern of PCH2:GFP together with ASY1:RFP in the male meiocytes of wildtype. Arrowheads indicate the chromosomal regions where the ASY1 removal was concomitant with the localization of PCH2. Scale bar: 10 μm.


( B ) Interaction assays of different ASY1 fragments ( with and without the closure motif ) .

(B) Interaction assays of different ASY1 fragments (with and without the closure motif).


( C ) Interaction analysis of the ASY1 closure motif ( ASY1571 - 596 ) with different ASY1 HORMA domain variants .

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(C) Interaction analysis of the ASY1 closure motif (ASY1571-596) with different ASY1 HORMA domain variants.


( A ) GalNAc - T expression levels in human liver ( The body map data for human liver was kindly provided by the " Gene Expression Applications research group at Illumina " , http : / / www . ebi . ac . uk / arrayexpress / experiments / E - MTAB - 513 / ) and HepG2 wild type cells quantified by RNAseq ( n = 2 , bars representing mean with SD ) .

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(A) GalNAc-T expression levels in human liver (The body map data for human liver was kindly provided by the "Gene Expression Applications research group at Illumina", http://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-513/) and HepG2 wild type cells quantified by RNAseq (n=2, bars representing mean with SD).


( B ) VVA lectin immunoblot of secretomes from HepG2 SC , SCΔT1 , SCΔT2 and SCΔT3 .

(B) VVA lectin immunoblot of secretomes from HepG2 SC, SCΔT1, SCΔT2 and SCΔT3.


Histograms showing the distribution of M / L quantitation ratios of monoglycosylated peptides identified in ( A ) HepG2SCΔT1 , ( B ) ΔT2 and ( C ) + T3 . Glycopeptides with M / L ratio < - 1 are colored red and glycopeptides with a M / L ratio > + 1 are colored green . ( D ) Venn diagram showing the distribution of candidates for isoform - specific sites among HepG2SCΔT1 , T2 and + T3 applying a log10 ( + / - 1 ) cut - off ( excluding sites identified in both TCL and SEC for each isoform ) , and ( E ) TCL alone and ( F ) SEC alone .

O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Histograms showing the distribution of M/L quantitation ratios of monoglycosylated peptides identified in (A) HepG2SCΔT1, (B) ΔT2 and (C) +T3. Glycopeptides with M/L ratio < -1 are colored red and glycopeptides with a M/L ratio > +1 are colored green. (D) Venn diagram showing the distribution of candidates for isoform-specific sites among HepG2SCΔT1, T2 and +T3 applying a log10 (+/-1) cut-off (excluding sites identified in both TCL and SEC for each isoform), and (E) TCL alone and (F) SEC alone.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with either no target cells ( NK ( - ) ) , autologous CD4 T cells ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) at an effector : target cell ratio of 1 : 2 . NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( A ) Representative histograms ( overlay ) displaying CD107a expression on NK cells after culture with the described culture conditions ( NK ( - ) , Mock , HIV ) . For the HIV - 1 co - culture condition , subsequent analyses of antigen expression were conducted by gating non - responsive ( CD107a - ) and responsive ( CD107a + ) NK cell subsets . ( B ) Relative frequency of CD107a + NK cells ( y - axis ) after culture in the described conditions ( x - axis ) ( n = 19 different donors ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

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Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (A) Representative histograms (overlay) displaying CD107a expression on NK cells after culture with the described culture conditions (NK(-), Mock, HIV). For the HIV-1 co-culture condition, subsequent analyses of antigen expression were conducted by gating non-responsive (CD107a-) and responsive (CD107a+) NK cell subsets. (B) Relative frequency of CD107a+ NK cells (y-axis) after culture in the described conditions (x-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with either no target cells ( NK ( - ) ) , autologous CD4 T cells ( Mock ) or enriched in vitro HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) at an effector : target cell ratio of 1 : 2 . NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry . ( C ) HIV - specific response of bulk NK cells displayed as percentage points ( p . p . ) CD107a + NK cells after normalization to mock CD4 T cells ( y - axis ) ( n = 19 different donors ) . Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points .

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Primary human NK cells (n = 19 different donors per condition) were co-incubated with either no target cells (NK(-)), autologous CD4 T cells (Mock) or enriched in vitro HIV-1-infected (NL4-3) CD4 T cells (HIV) at an effector:target cell ratio of 1:2. NK cell degranulation was quantified by measuring CD107a surface expression using flow cytometry. (C) HIV-specific response of bulk NK cells displayed as percentage points (p.p.) CD107a+ NK cells after normalization to mock CD4 T cells (y-axis) (n = 19 different donors). Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points.


Primary human NK cells ( n = 19 different donors per condition ) were co - incubated with HIV - 1 - infected ( NL4 - 3 ) CD4 T cells ( HIV ) ( E ) Representative histograms ( overlay ) showing TRAIL surface expression as fluorescence intensity on NK cells after co - incubation with HIV - 1 - infected CD4 T cells . Histograms show TRAIL expression for CD107a + and CD107a - NK cell subsets ( solid lines ) as well as their corresponding FMO controls ( dotted lines ) . Gate defining TRAIL + cells is indicated as well . ( F ) Left panel : Relative frequency of TRAIL + cells ( y - axis ) of CD107a + and CD107a - NK cell subsets ( x - axis ) after co - incubation with HIV - 1 - infected CD4 T cells . Right panel : Difference in relative frequency of TRAIL + cells ( y - axis ) between CD107a + and CD107a - cells displayed as p . p . ( n = 19 different donors ) . Data information : Wilcoxon signed - rank test . Adjustment for multiplicity : Benjamini and Hochberg false discovery rate ( FDR ) F , left panel : ) ; Bonferroni ( F , right panel Box plots represent the median and 25 % / 75 % percentile . Whiskers indicate the minimum and maximum data points . Lines connecting CD107a - and CD107a + cells refer to measured values of the same donor .

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Primary human NK cells (n = 19 different donors per condition) were co-incubated with HIV-1-infected (NL4-3) CD4 T cells (HIV) (E) Representative histograms (overlay) showing TRAIL surface expression as fluorescence intensity on NK cells after co-incubation with HIV-1-infected CD4 T cells. Histograms show TRAIL expression for CD107a+ and CD107a- NK cell subsets (solid lines) as well as their corresponding FMO controls (dotted lines). Gate defining TRAIL+ cells is indicated as well. (F) Left panel: Relative frequency of TRAIL+ cells (y-axis) of CD107a+ and CD107a- NK cell subsets (x-axis) after co-incubation with HIV-1-infected CD4 T cells. Right panel: Difference in relative frequency of TRAIL+ cells (y-axis) between CD107a+ and CD107a- cells displayed as p.p. (n = 19 different donors). Data information: Wilcoxon signed-rank test. Adjustment for multiplicity: Benjamini and Hochberg false discovery rate (FDR) F, left panel:); Bonferroni (F, right panel Box plots represent the median and 25%/75% percentile. Whiskers indicate the minimum and maximum data points. Lines connecting CD107a- and CD107a+ cells refer to measured values of the same donor.