Figure 3 ( A ) Real - time qPCR analysis of ANXA1 expression using mRNA extracted from the non - cancerous SB , cancerous 10C , and human CRC cell lines HCT116 , DLD1 and RKO , each grown to 50 % or 100 % confluency . All results were normalized to the ANXA1 mRNA levels in SB cells of 100 % confluency , which was designated as 1 . Data are mean values ± SEM . The experiment was performed in triplicates and repeated twice . * * p < 0 . 01 and * * * p < 0 . 001 ( student ' s t - test ) . ( B ) Confocal microscopy analysis of SB and 10C cells grown to 20 % ( top panels ) or 100 % ( bottom panels ) confluency followed by immuno - fluorescence staining of Annexin A1 ( green ) . The nuclei were stained with DAPI ( blue ) . A series of 20 - 50 consecutive images in the z - axis were stacked together to generate the 3 - D figure at 400x magnification . Annexin A1 is most abundantly expressed on the outer layer of 20 % confluent 10C ( pointed by arrows compared to 100 % confluent 10C , or SB of either confluency . Scale bars , x = 1 μm , y = 1 μm , z = 1 . 6 μm . The experiment was repeated at least twice . ( C ) Cell proliferation assay of adenoma - derived non - cancerous SB and cancerous 10C and human CRC cell lines HCT116 , DLD1 , SW480 , HT29 and RKO either untreated ( black lines ) or following treatment with control siRNA ( gray lines ) or ANXA1 - specific siRNA ( green lines ) . Data are mean values ± SEM . The experiment was performed in triplicates and repeated three times . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 , compared to the untreated cells ( two - way ANOVA ) . ( D ) Cell proliferation assay of SB ( left panel ) and RKO ( right panel ) cells transfected with ANXA1 ( red line ) , as compared to the control cells ( black lines ) . Data are mean values ± SEM . The experiment was performed in triplicates and repeated three times . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( two - way ANOVA ) . ( E ) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1 - specific siRNA . The tumor volumes were measured after 7 days post injection ( left panel , n = 4 ) . For each mouse , the tumor resulting from ANXA1 - specific siRNA treated cells was normalized to that from control siRNA - treated cells , which was designated 100 % . The line represents the average . * p < 0 . 05 ( paired t - test ) . The individual tumor pairs are shown on the right panel : top , tumors arising from control siRNA treated cells ; bottom , tumors arising from ANXA1 - specific siRNA treated cells .

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

Figure 3(A) Real-time qPCR analysis of ANXA1 expression using mRNA extracted from the non-cancerous SB, cancerous 10C, and human CRC cell lines HCT116, DLD1 and RKO, each grown to 50% or 100% confluency. All results were normalized to the ANXA1 mRNA levels in SB cells of 100% confluency, which was designated as 1. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. **p<0.01 and ***p<0.001 (student's t-test).(B) Confocal microscopy analysis of SB and 10C cells grown to 20% (top panels) or 100% (bottom panels) confluency followed by immuno-fluorescence staining of Annexin A1 (green). The nuclei were stained with DAPI (blue). A series of 20-50 consecutive images in the z-axis were stacked together to generate the 3-D figure at 400x magnification. Annexin A1 is most abundantly expressed on the outer layer of 20% confluent 10C (pointed by arrows compared to 100% confluent 10C, or SB of either confluency. Scale bars, x = 1 μm, y = 1 μm, z = 1.6 μm. The experiment was repeated at least twice.(C) Cell proliferation assay of adenoma-derived non-cancerous SB and cancerous 10C and human CRC cell lines HCT116, DLD1, SW480, HT29 and RKO either untreated (black lines) or following treatment with control siRNA (gray lines) or ANXA1-specific siRNA (green lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, ** p<0.01 and *** p<0.001, compared to the untreated cells (two-way ANOVA).(D) Cell proliferation assay of SB (left panel) and RKO (right panel) cells transfected with ANXA1 (red line), as compared to the control cells (black lines). Data are mean values ± SEM. The experiment was performed in triplicates and repeated three times. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).(E) Xenografted tumor growth in nude mice following subcutaneous and bilateral inoculation of HCT116 cells transfected with control siRNA or ANXA1-specific siRNA. The tumor volumes were measured after 7 days post injection (left panel, n=4). For each mouse, the tumor resulting from ANXA1-specific siRNA treated cells was normalized to that from control siRNA-treated cells, which was designated 100%. The line represents the average. *p<0.05 (paired t-test). The individual tumor pairs are shown on the right panel: top, tumors arising from control siRNA treated cells; bottom, tumors arising from ANXA1-specific siRNA treated cells.


Figure 4 ( A ) Real - time qPCR analysis of ANXA1 mRNA levels in SB , 10C , HCT116 and DLD1 cells incubated with wild - type F . nucleatum 12230 ( Fn ) or fadA - deletion mutant US1 ( US1 ) at MOI of 50 : 1 for the indicated time periods . The results were normalized to those obtained with untreated cells and were the mean of 3 independent experiments each performed in triplicates . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ( two - way ANOVA ) . ( B ) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated , or incubated with BSA ( 1000 µg / ml ) , or mFadA ( 1000 µg / ml ) , or FadAc ( 100 , 300 , or 1000 µg / ml ) for 1 hr . Data are mean values ± SD . The experiment was performed in triplicates and repeated more than three times . * * p < 0 . 001 ( one - way ANOVA ) . ( C ) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA ( dotted black line ) or CDH1 - specific siRNA ( dotted red line ) followed by no treatment , or incubation with BSA ( 1000 µg / ml ) or FadAc ( 1000 µg / ml ) for 1 hr . C , untreated control . Data are mean values ± SD . The experiment was performed in triplicates and repeated twice . * * * p < 0 . 001 ( two - way ANOVA ) . ( D ) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE - labeled F . nucleatum 12230 ( Fn ) for 1 hr at MOI of 5 : 1 . Annexin A1 was stained green and E - cadherin blue . Imagines are 800x magnification . Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass . The experiment was repeated three times . Scale bar , 250 nm .

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

Figure 4(A) Real-time qPCR analysis of ANXA1 mRNA levels in SB, 10C, HCT116 and DLD1 cells incubated with wild-type F. nucleatum 12230 (Fn) or fadA-deletion mutant US1 (US1) at MOI of 50:1 for the indicated time periods. The results were normalized to those obtained with untreated cells and were the mean of 3 independent experiments each performed in triplicates. *p<0.05, **p<0.01 and ***p<0.001 (two-way ANOVA).(B) Flow cytometry analysis of Annexin A1 expression in 10C cells either untreated, or incubated with BSA (1000 µg/ml), or mFadA (1000 µg/ml), or FadAc (100, 300, or 1000 µg/ml) for 1 hr. Data are mean values ± SD. The experiment was performed in triplicates and repeated more than three times. **p<0.001 (one-way ANOVA).(C) Flow cytometry analysis of Annexin A1 in 10C cells transfected with control siRNA (dotted black line) or CDH1-specific siRNA (dotted red line) followed by no treatment, or incubation with BSA (1000 µg/ml) or FadAc (1000 µg/ml) for 1 hr. C, untreated control. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. ***p<0.001 (two-way ANOVA).(D) Confocal microscopy analysis of SB and 10C cells either untreated or following incubation with CFSE-labeled F. nucleatum 12230 (Fn) for 1 hr at MOI of 5:1. Annexin A1 was stained green and E-cadherin blue. Imagines are 800x magnification. Note the enhanced expression of Annexin A1 in 10C compared to SB and its location on the outer layer of the cell mass. The experiment was repeated three times. Scale bar, 250 nm.


Figure 5 ( A ) Western blotting analysis of FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in DLD1 cells following incubation with FadAc for 15 or 120 min . C , untreated cells . β - actin was included as an internal control . The experiment was repeated three times . ( B ) Co - immunoprecipitation with Annexin A1 . DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti - Annexin A1 polyclonal antibody ( α - Annexin A1 ) or control rabbit IgG . FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in the eluates were detected by Western blot . C , untreated control . The experiment was repeated three times . ( C ) Co - immunoprecipiation with FadA . DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with mouse anti - FadA monoclonal antibody ( α - FadA ) or control mouse IgG . FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) and β - catenin in the eluates were detected by Western blot . The experiment was repeated three times . ( D ) Confocal microscopy analysis of 10C cells either untreated ( top panel ) or following incubation with CFSE - labeled F . nucleatum 12230 ( red , bottom panel ) for 3 hrs and then immunofluorescent - stained for Annexin A1 ( green ) and E - cadherin ( blue ) . Images are 1200x magnification . A side view of the enlarged image is shown on the far right . Note the enhanced expression of Annexin A1 in the F . nucleatum - bound cells and the co - localization of Annexin A1 , E - cadherin and F . nucleatum on the cell membranes ( arrows ) . The experiment was repeated more than three times . Scale bar , 500 nm . ( E ) Confocal microscopy analysis of 10C cells following incubation with Alexa Fluor ™ 488 - conjugated BSA , mFadA , or FadAc ( red ) for 1 hr and immuno - staining of Annexin A1 ( green ) and E - cadherin ( blue ) . Images are 1200x magnification . Note the enhanced expression of Annexin A1 and its co - localization with E - cadherin in the presence of FadAc ( arrows ) , but not with BSA and mFadA . The experiment was repeated twice . The side views are shown to the right and bottom of each image . Scale bar , 500 nm .

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

Figure 5(A) Western blotting analysis of FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in DLD1 cells following incubation with FadAc for 15 or 120 min. C, untreated cells. β-actin was included as an internal control. The experiment was repeated three times.(B) Co-immunoprecipitation with Annexin A1. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with rabbit anti-Annexin A1 polyclonal antibody (α-Annexin A1) or control rabbit IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. C, untreated control. The experiment was repeated three times.(C) Co-immunoprecipiation with FadA. DLD1 cell lysates were incubated with FadAc for 15 or 120 min and mixed with agarose beads conjugated with mouse anti-FadA monoclonal antibody (α-FadA) or control mouse IgG. FadA, E-cadherin (CDH1), Annexin A1 (ANXA1) and β-catenin in the eluates were detected by Western blot. The experiment was repeated three times.(D) Confocal microscopy analysis of 10C cells either untreated (top panel) or following incubation with CFSE-labeled F. nucleatum 12230 (red, bottom panel) for 3 hrs and then immunofluorescent-stained for Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. A side view of the enlarged image is shown on the far right. Note the enhanced expression of Annexin A1 in the F. nucleatum-bound cells and the co-localization of Annexin A1, E-cadherin and F. nucleatum on the cell membranes (arrows). The experiment was repeated more than three times. Scale bar, 500 nm.(E) Confocal microscopy analysis of 10C cells following incubation with Alexa Fluor™ 488-conjugated BSA, mFadA, or FadAc (red) for 1 hr and immuno-staining of Annexin A1 (green) and E-cadherin (blue). Images are 1200x magnification. Note the enhanced expression of Annexin A1 and its co-localization with E-cadherin in the presence of FadAc (arrows), but not with BSA and mFadA. The experiment was repeated twice. The side views are shown to the right and bottom of each image. Scale bar, 500 nm.


Figure 6Flow cytometry analysis of SB ( A ) , 10C ( B ) , DLD1 ( C ) and HCT116 ( D ) cells incubated with CFSE - labeled F . nucleatum 12230 ( Fn ) or its fadA - deletion mutant US1 ( US1 ) at MOI of 10 - 20 : 1 for the indicated time and immuno - stained with anti - Annexin A1 antibodies . Shown on the top panels are the density plots . x - axis , CFSE - labeled F . nucleatum or US1 ( CFSE - Fn ) ; y - axis , Annexin A1 ( ANXA1 ) . Shown on the bottom panels are the percentages of Annexin A1 - positive ( solid bars ) or negative ( clear bars ) cells bound by F . nucleatum or US1 out of the total number of cells analyzed . Data are mean values ± SD . The experiments were performed in triplicates and repeated 2 - 3 times . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) .

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

Figure 6Flow cytometry analysis of SB (A), 10C (B), DLD1 (C) and HCT116 (D) cells incubated with CFSE-labeled F. nucleatum 12230 (Fn) or its fadA-deletion mutant US1 (US1) at MOI of 10-20:1 for the indicated time and immuno-stained with anti-Annexin A1 antibodies. Shown on the top panels are the density plots. x-axis, CFSE-labeled F. nucleatum or US1 (CFSE-Fn); y-axis, Annexin A1 (ANXA1). Shown on the bottom panels are the percentages of Annexin A1-positive (solid bars) or negative (clear bars) cells bound by F. nucleatum or US1 out of the total number of cells analyzed. Data are mean values ± SD. The experiments were performed in triplicates and repeated 2-3 times. *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA).


Figure 7 ( A ) Colorectal tumors generated in Apcmin / + mice following treatment with PBS , E . coli DH5α ( E . coli ) , fadA - deletion mutant US1 ( US1 ) or F . nucleatum 12230 ( Fn ) . Each symbol represents one mouse . Horizontal lines represent mean values . Representative tumors formed in the mouse colon are shown on the right , pointed by blue arrows on the right . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA ) . ( B ) ANXA1 mRNA levels in Apcmin / + mouse colonic tumors and normal colonic tissues as measured by real - time qPCR . Each symbol represents one mouse . Horizontal lines represent mean values . * p < 0 . 05 , * * p < 0 . 01 ( two - way ANOVA ) . ( C ) Positive correlation between fadA gene copy numbers ( x - axis ) and ANXA1 mRNA levels ( y - axis ) in F . nucleatum - induced Apcmin / + mouse colonic tumors ( n = 34 ; Pearson ' s correlation ) . Each dot represents the average of qPCR results performed in duplicates ( D ) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients ( n = 18 ) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR . Each symbol shows the average of duplicate qPCR results from one patient . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - test ) . ( E ) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients ( n = 18 ) . Each symbol shows the average of duplicate qPCR results from one patient . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - test ) . ( F ) Positive correlation between fadA gene copy numbers ( x - axis ) and ANXA1 mRNA levels ( y - axis ) in human colorectal adenocarcinoma tissues ( n = 18 ; Pearson ' s correlation ) . Each dot represents the average of qPCR results performed in duplicates . ( G ) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients . The frozen sections were incubated with rabbit anti - AnnexinA1 polyclonal antibodies and 5G11 mouse anti - FadA monoclonal antibodies . The slides were then stained with Alexa Fluor ® 680 - conjugated donkey anti - rabbit and Alexa Fluor ® 555 - conjugated goat anti - mouse , washed , and covered in mounting medium containing DAPI . The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas . Scale bar , 50 μm . Co - localization of FadA ( red ) and Annexin A1 ( green ) was observed in carcinomas but not in the paired normal tissues .

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

Figure 7(A) Colorectal tumors generated in Apcmin/+ mice following treatment with PBS, E. coli DH5α (E. coli), fadA-deletion mutant US1 (US1) or F. nucleatum 12230 (Fn). Each symbol represents one mouse. Horizontal lines represent mean values. Representative tumors formed in the mouse colon are shown on the right, pointed by blue arrows on the right. *p<0.05, **p<0.01 (one-way ANOVA).(B) ANXA1 mRNA levels in Apcmin/+ mouse colonic tumors and normal colonic tissues as measured by real-time qPCR. Each symbol represents one mouse. Horizontal lines represent mean values. *p<0.05, **p<0.01 (two-way ANOVA).(C) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y- axis) in F. nucleatum-induced Apcmin/+ mouse colonic tumors (n=34; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates(D) Abundance of fadA in the paired normal and adenocarcinoma tissues from human CRC patients (n=18) expressed as ratio of fadA over total 16S rRNA genes determined by qPCR. Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).(E) ANXA1 mRNA levels in paired normal and adenocarcinoma tissues from CRC patients (n=18). Each symbol shows the average of duplicate qPCR results from one patient. Horizontal lines represent mean values. **p<0.01 (paired t-test).(F) Positive correlation between fadA gene copy numbers (x-axis) and ANXA1 mRNA levels (y-axis) in human colorectal adenocarcinoma tissues (n=18; Pearson's correlation). Each dot represents the average of qPCR results performed in duplicates.(G) Confocal microscopy analysis of paired normal and carcinoma tissues from two colon cancer patients. The frozen sections were incubated with rabbit anti-AnnexinA1 polyclonal antibodies and 5G11 mouse anti-FadA monoclonal antibodies. The slides were then stained with Alexa Fluor®680-conjugated donkey anti-rabbit and Alexa Fluor®555-conjugated goat anti-mouse, washed, and covered in mounting medium containing DAPI. The scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for the normal tissues and 400x for the carcinomas. Scale bar, 50 μm. Co-localization of FadA (red) and Annexin A1 (green) was observed in carcinomas but not in the paired normal tissues.


Figure 8 ( A ) Flow cytometry analysis of β - catenin expression in 10C , HCT116 and DLD1 cells following transfection with control siRNA ( clear bars ) or ANXA1 - specific siRNA ( solid bars ) and incubation with F . nucleatum 12230 at MOI of ~ 20 : 1 for indicated time periods . The geometric means of cells treated with control siRNA at time 0 was designated as 1 . Data are mean values ± SD . The experiment was performed in duplicates or triplicates and repeated 1 - 3 times . * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) . ( B ) Immuno - staining of β - catenin in 10C cells following transfection with control siRNA or ANXA1 - specific siRNA and incubation with F . nucleatum 12230 ( Fn ) at MOI of ~ 100 : 1 for 2 hrs . β - catenin was stained with Alexa Fluor ® 680 ( red ) and the nuclei with DAPI ( blue ) . The images were captured with confocal microscope at 800x magnification . - , no bacteria added . Note the increased expression of β - catenin and its nucleus translocation in response to F . nucleatum in control siRNA - treated cells , but not in ANXA1 siRNA - treated cells . The experiment was repeated twice . Scale bar , 200 nm . ( C ) Real - time qPCR analysis of CCND1 expression using mRNA extracted from the cancerous 10C , and human CRC cells HCT116 and RKO , each grown to 50 % or 100 % confluency . Data are mean values ± SEM . The experiment was performed in triplicates and repeated twice . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - test ) . ( D ) Western - blot analysis of Cyclin D1 , Annexin A1 , and β - actin in RKO cells transfected with control vector or ANXA1 . Induction of Cyclin D1 was observed in response to increased Annexin A1 . The experiment was repeated twice .

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

Figure 8(A) Flow cytometry analysis of β-catenin expression in 10C, HCT116 and DLD1 cells following transfection with control siRNA (clear bars) or ANXA1-specific siRNA (solid bars) and incubation with F. nucleatum 12230 at MOI of ~20:1 for indicated time periods. The geometric means of cells treated with control siRNA at time 0 was designated as 1. Data are mean values ± SD. The experiment was performed in duplicates or triplicates and repeated 1-3 times. **p<0.01, *** p<0.001 (two-way ANOVA).(B) Immuno-staining of β-catenin in 10C cells following transfection with control siRNA or ANXA1-specific siRNA and incubation with F. nucleatum 12230 (Fn) at MOI of ~100:1 for 2 hrs. β-catenin was stained with Alexa Fluor®680 (red) and the nuclei with DAPI (blue). The images were captured with confocal microscope at 800x magnification. -, no bacteria added. Note the increased expression of β-catenin and its nucleus translocation in response to F. nucleatum in control siRNA-treated cells, but not in ANXA1 siRNA-treated cells. The experiment was repeated twice. Scale bar, 200 nm.(C) Real-time qPCR analysis of CCND1 expression using mRNA extracted from the cancerous 10C, and human CRC cells HCT116 and RKO, each grown to 50% or 100% confluency. Data are mean values ± SEM. The experiment was performed in triplicates and repeated twice. *p<0.05, ***p<0.001 (student's t-test).(D) Western-blot analysis of Cyclin D1, Annexin A1, and β-actin in RKO cells transfected with control vector or ANXA1. Induction of Cyclin D1 was observed in response to increased Annexin A1. The experiment was repeated twice.


Figure 1 ( A ) Tru - Seq RNA sequence analysis of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) and posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in the midbrain FP , together with Th and Pitx3 . Lower levels of Pbx3 are also expressed in the mFP . Gdf7 and Wnt1 are restricted to the mRP at E12 . 5 . ( B ) Pbx1 is expressed in the intermediate zone ( IZ ) and marginal zones ( MZ ) , but not the ventricular zone ( VZ ) , of the mFP at E12 . 5 , as detected by in situ hybridization . ( C ) PBX1 is first detected in the ventro - lateral part of the LMX1a + domain at E10 , preceding the birth of the first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in all mDA neurons , but not all PBX1 + cells are TH + . White boxes indicate the area showed in higher magnification ( right ) . ( D ) At E11 . 5 , PBX1 protein defines a subpopulation of NURR1 + neuroblasts and labels all NURR1 + TH + mDA neurons . ( E ) PBX1 co - localizes with PITX3 and is also detected in a subpopulation of NURR1 + PITX3 - postmitotic neuroblasts at E12 . 5 . Higher magnification revealed three different populations of postmitotic cells : primary neuroblasts ( NURR1 + PBX1A - PITX3 - cells , green ) , secondary neuroblasts ( NURR1 + PBX1A + PITX3 - cells , yellow / orange ) and tertiary neuroblasts / mDA neurons ( NURR1 + PBX1A + PITX3 + cells , white ) . Nuclear staining , Dapi ( 4 ′ , 6 - diamidino - 2 - phenylindole , blue ) . All scale bars , 20µm .

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Figure 1(A) Tru-Seq RNA sequence analysis of E12.5 midbrain floor-plate (mFP), midbrain roof-plate (mRP), anterior (A, adjacent anterior FP) and posterior (P, adjacent posterior FP). Pbx1 is enriched in the midbrain FP, together with Th and Pitx3. Lower levels of Pbx3 are also expressed in the mFP. Gdf7 and Wnt1 are restricted to the mRP at E12.5.(B) Pbx1 is expressed in the intermediate zone (IZ) and marginal zones (MZ), but not the ventricular zone (VZ), of the mFP at E12.5, as detected by in situ hybridization.(C) PBX1 is first detected in the ventro-lateral part of the LMX1a+ domain at E10, preceding the birth of the first (TH+) mDA neurons at E10.5. At E12.5, PBX1 is present in all mDA neurons, but not all PBX1+ cells are TH+. White boxes indicate the area showed in higher magnification (right).(D) At E11.5, PBX1 protein defines a subpopulation of NURR1+ neuroblasts and labels all NURR1+TH+ mDA neurons.(E) PBX1 co-localizes with PITX3 and is also detected in a subpopulation of NURR1+PITX3- postmitotic neuroblasts at E12.5. Higher magnification revealed three different populations of postmitotic cells: primary neuroblasts (NURR1+PBX1A-PITX3-cells, green), secondary neuroblasts (NURR1+PBX1A+PITX3-cells, yellow/orange) and tertiary neuroblasts/mDA neurons (NURR1+PBX1A+PITX3+cells, white). Nuclear staining, Dapi (4′,6-diamidino-2-phenylindole, blue). All scale bars, 20µm.


Figure 2 ( A ) PBX1A is the isoform detected in the TH + NURR1 + mDA neurons at E12 . 5 . ( C ) Immunofluorescence microscopy analysis of 7 - week old human VM tissue showing that all mDAn are positive for PBX1 , suggesting a conserved role for this factor in the development of these neurons in mice and humans . ( D ) PBX1 is also present in TH + adult mDA neurons of the substantia nigra ( SN ) and ventral tegmental area ( VTA ) .

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Figure 2(A) PBX1A is the isoform detected in the TH+NURR1+mDA neurons at E12.5.(C) Immunofluorescence microscopy analysis of 7-week old human VM tissue showing that all mDAn are positive for PBX1, suggesting a conserved role for this factor in the development of these neurons in mice and humans.(D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA).


Figure 3 ( A ) To the left , representative immunofluorescence microscopy / confocal images showing that the number of TH + cells is reduced in Pbx1 - / - embryos . Deletion of Pbx1 decreases the proportion of NURR1 + neuroblasts that become TH + neurons at E12 . 5 , indicating that Pbx1 is required for the proper maturation of NURR1 + neuroblasts . To the right , quantification of these experiments . ( B ) Ectopic PBX3 is detected in all rostro - caudal levels of the VM in Pbx1 - / - mice at E12 . 5 , indicating a repressive function of PBX1 on Pbx3 expression . ( C ) Representative immunofluorescence microscopy / confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 ( cKO , Pbx1flox / flox ; Pbx3 - / - ; Th - IRES - Cre - ERT ) reduces the number of PITX3 + cells in rostral VM levels , but not of NURR1 + cells , when compared to double heterozygous at E14 . 5 ( cHET , Pbx1flox / + ; Pbx3 - / + ; Th - IRES - Cre - ERT ) . White boxes are magnified to the right . ( D ) At E18 . 5 , a drastic reduction of TH + , PITX3 + and NURR1 + cells is detected from anterior to posterior levels in cKO embryos , but not in cHET . Pyknotic NURR1 + nuclei are observed in cKO embryos ( arrowheads ) . White boxes are magnified to the right . ( E ) The total number of TH + mDA neurons is dramatically reduced and there is an increase in aCASP3 + cells in cKO embryos compared to cHET mice at E18 . 5 .

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Figure 3(A) To the left, representative immunofluorescence microscopy/confocal images showing that the number of TH+cells is reduced in Pbx1-/- embryos. Deletion of Pbx1 decreases the proportion of NURR1+neuroblasts that become TH+neurons at E12.5, indicating that Pbx1 is required for the proper maturation of NURR1+neuroblasts. To the right, quantification of these experiments.(B) Ectopic PBX3 is detected in all rostro-caudal levels of the VM in Pbx1-/- mice at E12.5, indicating a repressive function of PBX1 on Pbx3 expression.(C) Representative immunofluorescence microscopy/confocal images and quantification showing that the conditional deletion of Pbx1 and Pbx3 (cKO, Pbx1flox/flox;Pbx3-/-;Th-IRES-Cre-ERT) reduces the number of PITX3+cells in rostral VM levels, but not of NURR1+cells, when compared to double heterozygous at E14.5 (cHET, Pbx1flox/+;Pbx3-/+;Th-IRES-Cre-ERT). White boxes are magnified to the right.(D) At E18.5, a drastic reduction of TH+, PITX3+ and NURR1+cells is detected from anterior to posterior levels in cKO embryos, but not in cHET. Pyknotic NURR1+nuclei are observed in cKO embryos (arrowheads). White boxes are magnified to the right.(E) The total number of TH+ mDA neurons is dramatically reduced and there is an increase in aCASP3+ cells in cKO embryos compared to cHET mice at E18.5.


Figure 4 ( B ) Representative immunofluorescence microscopy / confocalimages showing that PBX1 - overexpression increases the number of TH + neurons after 8 days of differentiation of hNES cells ( AF22 line derived from induced pluripotent stem cells ) . ( C ) While the number of cells ( Dapi + ) in both conditions did not change , the number of TH + cells increased 2 . 3 fold in PBX1 - overexpressing cells . ( D ) TH + cells derived from the PBX1 - overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3 . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates per condition and p - value ( T - test ) are indicated in the graphs .

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Figure 4(B) Representative immunofluorescence microscopy/confocalimages showing that PBX1-overexpression increases the number of TH+neurons after 8 days of differentiation of hNES cells (AF22 line derived from induced pluripotent stem cells). (C) While the number of cells (Dapi+) in both conditions did not change, the number of TH+cells increased 2.3 fold in PBX1-overexpressing cells.(D) TH+cells derived from the PBX1-overexpressing hNES cells were also positive for mDAn markers such as LMX1A and PITX3. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates per condition and p-value (T-test) are indicated in the graphs.


Figure 5 ( B ) Schematic of the track of PBX1 ChIP - seq compared to IgGChIP - seq as a control on different loci with peaks identified by HOMER . Each locus represents a 20kb chromosome region . ( C ) List of the 29 most regulated genes ( t - test q - value < 0 . 05 ) identified by Gene Set Enrichment Analysis ( GSEA ) and ranked by p - value . RPKM stands for Reads Per Kilobase of transcript per Million mapped reads . GSEA was performed on genes identified by RNA - Seq as differentially expressed in the VM at E12 . 5 compared to adjacent brain regions and identified by PBX1Chip - Seq in proximity to transcription starting sites . ( D ) Graphic representation of these top 29 genes ( red dots ) in the GSEA curve showing a high correlation between the cistrome and transcriptome ( p - value < 0 . 001 , q - value < 0 . 005 ) . ( E ) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 ( Pitx3 - Gbf1 ) , Onecut2and Nfe2l1 genes . No co - immunoprecipitation was detected for the unrelated gene , Lrp1b ( Low Density Lipoprotein Receptor - Related Protein 1B ) . ( F , G ) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1 - / - ; Pbx3 + / - mutant embryos at E12 . 5 , compared to wild - type ( Pbx1 + / + ) littermates . ( F ) Representative immunofluorescence microscopy / confocal images showing that decreased levels of PITX3 and number of PITX3 + cells in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . This reduction is more evident in the basal plate ( BP ) and in the midline ( M ) , as shown at higher magnification ( right ) . ( G ) Double ONECUT2 + TH + cells were detected only in the lateral part of the mFP in E12 . 5 Pbx1 - / - ; Pbx3 + / - embryos . Selected areas are magnified to the right . A scheme ( right ) shows the distribution of cells in Pbx1 + / + and Pbx1 - / - ; Pbx3 + / - embryos . All scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates ( dots ) per condition and p - value ( T - test ) are indicated in each graph .

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Figure 5(B) Schematic of the track of PBX1 ChIP-seq compared to IgGChIP-seq as a control on different loci with peaks identified by HOMER. Each locus represents a 20kb chromosome region.(C) List of the 29 most regulated genes (t-test q-value <0.05) identified by Gene Set Enrichment Analysis (GSEA) and ranked by p-value. RPKM stands for Reads Per Kilobase of transcript per Million mapped reads. GSEA was performed on genes identified by RNA-Seq as differentially expressed in the VM at E12.5 compared to adjacent brain regions and identified by PBX1Chip-Seq in proximity to transcription starting sites.(D) Graphic representation of these top 29 genes (red dots) in the GSEA curve showing a high correlation between the cistrome and transcriptome (p-value <0.001, q-value<0.005).(E) ChIP followed by PCR confirmed that PBX1 binds to genomic regions in close proximity to the TSS of Pitx3 (Pitx3-Gbf1), Onecut2and Nfe2l1 genes. No co-immunoprecipitation was detected for the unrelated gene, Lrp1b (Low Density Lipoprotein Receptor-Related Protein 1B).(F, G) The regulation of Pitx3 and Onecut2 by PBX1 was confirmed in Pbx1-/-;Pbx3+/- mutant embryos at E12.5, compared to wild-type (Pbx1+/+) littermates. (F) Representative immunofluorescence microscopy/confocal images showing that decreased levels of PITX3 and number of PITX3+cells in E12.5 Pbx1-/-;Pbx3+/- embryos. This reduction is more evident in the basal plate (BP) and in the midline (M), as shown at higher magnification (right). (G) Double ONECUT2+TH+cells were detected only in the lateral part of the mFP in E12.5 Pbx1-/-;Pbx3+/- embryos. Selected areas are magnified to the right. A scheme (right) shows the distribution of cells in Pbx1+/+ and Pbx1-/-;Pbx3+/- embryos. All scale bars, 40µm. Error bars represent mean ± standard deviation. Biological replicates (dots) per condition and p-value (T-test) are indicated in each graph.


Figure 6 ( A ) Representative immunofluorescence microscopy / confocal images showing that NFE2L1 is detected in TH + mDAn and neuroblasts at E12 . 5 . ( B ) The expression of NFE2L1 in TH + neurons is dramatically reduced in Pbx1 - / - ; Pbx3 + / - compared to Pbx1 + / + embryos at E12 . 5 ( C ) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2 - treated ( 100µm for 12 hours ) TH + neurons derived from hNES cells differentiated for 8 days ( percentage ) . White arrows indicate double TH + aCASP3 + neurons . ( D ) PBX1 overexpression protected TH + neurons derived from hNES cells from the oxidative stress induced by H2O2 ( 100µm for 12 hours ) , resulting in reduced levels of aCASP3 in TH + neurons ( percentage ) . ( E ) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining ( dark - blue / black , indicated by arrowheads ) in the nuclei of neuromelanin + cells ( NM + , cytosolic brown aggregates ) of the SN . In contrast , PBX1 staining was severely reduced in PD patients . ( F ) Graphical representation of PBX1 intensity levels in the SN of NM + neurons in all individual controls and PD patients analyzed . Each dot in the graph represents the level of PBX1 in the nuclei of one single NM + neuron . ( G ) Strong immunoreactivity of the PBX1 direct target , NFE2L1 , was also detected as very well defined nuclear punctuate structures in NM + cells of the SN in control samples ( arrowheads ) , but were dramatically reduced in PD patients . Yellow asterisks indicate the nucleoli . ( H ) Percentage of NM + neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients . ( I ) Graphical representation of all individual controls and PD patients analyzed , showing that NM + neurons with NFE2L1 + nuclei in the SN of PD patients are less than 10 % or are totally absent .

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Figure 6(A) Representative immunofluorescence microscopy/confocal images showing that NFE2L1 is detected in TH+mDAn and neuroblasts at E12.5.(B) The expression of NFE2L1 in TH+neurons is dramatically reduced in Pbx1-/-;Pbx3+/- compared to Pbx1+/+ embryos at E12.5(C) shRNA lentiviruses against NFE2L1 increased the levels of aCASP3 in H2O2-treated (100µm for 12 hours) TH+neurons derived from hNES cells differentiated for 8 days (percentage). White arrows indicate double TH+aCASP3+neurons.(D) PBX1 overexpression protected TH+neurons derived from hNES cells from the oxidative stress induced by H2O2 (100µm for 12 hours), resulting in reduced levels of aCASP3 in TH+neurons (percentage).(E) Human postmortem SN tissue of control donors exhibited PBX1 chromogenic staining (dark-blue/black, indicated by arrowheads) in the nuclei of neuromelanin+cells (NM+, cytosolic brown aggregates) of the SN. In contrast, PBX1 staining was severely reduced in PD patients. (F) Graphical representation of PBX1 intensity levels in the SN of NM+ neurons in all individual controls and PD patients analyzed. Each dot in the graph represents the level of PBX1 in the nuclei of one single NM+neuron.(G) Strong immunoreactivity of the PBX1 direct target, NFE2L1, was also detected as very well defined nuclear punctuate structures in NM+cells of the SN in control samples (arrowheads), but were dramatically reduced in PD patients. Yellow asterisks indicate the nucleoli. (H) Percentage of NM+ neurons showing nuclear NFE2L1 staining in the SN of control donors and PD patients. (I) Graphical representation of all individual controls and PD patients analyzed, showing that NM+neurons with NFE2L1+nuclei in the SN of PD patients are less than 10% or are totally absent.


Figure 1A Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data ( No . GSE14905 ) . B Expression of ptpn11 in human PBMCs of psoriatic patients ( n = 14 ) and normal controls ( n = 16 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . C Representative SHP2 staining in skin sections of psoriatic patients ( n = 13 ) and normal controls ( n = 5 ) . Scale bars : 200 μm . D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls . E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients ( n = 25 ) and normal controls ( n = 25 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . F Representative p - ERK staining of skin sections of psoriatic patients and normal controls . Scale bars : 200 μm . G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 ( n = 6 / group ) . Data were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . H Representative histological sections of IMQ - treated or non - treated dorsal back of C57BL / 6 mice at day 5 . Scale bar : 100 μm . I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients ( n = 20 ) and normal healthy controls ( n = 20 ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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Figure 1A Expression of PTPN11 (gene encoding SHP2) in skin lesions in psoriatic patients compared with skin from healthy donors based on microarray data (No. GSE14905). B Expression of ptpn11 in human PBMCs of psoriatic patients (n=14) and normal controls (n=16). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.C Representative SHP2 staining in skin sections of psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm.D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls.E The catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic patients (n=25) and normal controls (n=25). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.F Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 μm.G Quantitative PCR analysis of Ptpn11 mRNA levels in the IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5 (n=6/group). Data were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.H Representative histological sections of IMQ-treated or non-treated dorsal back of C57BL/6 mice at day 5. Scale bar: 100 μm.I Quantitative PCR analysis of mRNA levels in human PBMCs derived from psoriatic patients (n=20) and normal healthy controls (n=20). Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Figure 2C57BL / 6 mice ( n = 6 / group ) were treated with indicated dose of SHP099 or vehicle for 4 days . A Phenotypic presentation ( top ) , H & E staining ( middle ) and statistical data ( bottom ) ( mean ± SEM ) of dorsal skin . Scale bar : 100 μm . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 . ELISA quantification of protein levels of cytokines in mouse serum . Quantitative PCR analysis of mRNA encoding psoriasis - related cytokines in the dorsal skin of C57BL / 6 mice treated with indicated dose of SHP099 or vehicle for 4 days . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * P < 0 . 05 , * * P < 0 . 01 .

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Figure 2C57BL/6 mice (n=6/group) were treated with indicated dose of SHP099 or vehicle for 4 days. A Phenotypic presentation (top), H&E staining (middle) and statistical data (bottom) (mean ± SEM) of dorsal skin. Scale bar: 100 μm. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.ELISA quantification of protein levels of cytokines in mouse serum. Quantitative PCR analysis of mRNA encoding psoriasis-related cytokines in the dorsal skin of C57BL/6 mice treated with indicated dose of SHP099 or vehicle for 4 days. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test *P<0.05, **P<0.01.


Figure 3Violin plot showed the relative expression of 47 inflammation - associated genes based on sample groups and cell clusters .

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Figure 3Violin plot showed the relative expression of 47 inflammation-associated genes based on sample groups and cell clusters.


Figure 4D Gene set enrichment analysis ( GSEA ) of the NF - κB signaling pathway . E Volcano plots of DEGs that were upregulated ( red ) or downregulated ( blue ) in myeloid cells . Examples of DEGs were labeled . G Expression patterns of the top ten hub genes in ( F ) . Heatmap ( top ) displays the average expression in the three groups and in different myeloid cell subsets . Boxplots ( bottom ) show the corresponding expression distribution .

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Figure 4D Gene set enrichment analysis (GSEA) of the NF-κB signaling pathway.E Volcano plots of DEGs that were upregulated (red) or downregulated (blue) in myeloid cells. Examples of DEGs were labeled.G Expression patterns of the top ten hub genes in (F). Heatmap (top) displays the average expression in the three groups and in different myeloid cell subsets. Boxplots (bottom) show the corresponding expression distribution.


Figure 5Representative images ( A and E ) , histological sections of dorsal back ( B and F ) and clinical scores ( C and G ) and quantitative PCR analysis of mRNA levels in dorsal skin ( D and H ) from wild - type ( n = 6 ) and M - Shp2 - / - mice or DC - Shp2 - / - mice ( n = 6 ) treated with IMQ for 4 days . Scale bar : 100 μm . Left : H & E ( hematoxylin and eosin ) staining data ; right : statistical data ( mean ± SEM ) . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values were calculated by Tukey multiple comparison test ( B , D , F , H ) or Bonferroni multiple comparison test ( C , G ) . * P < 0 . 05 , * * P < 0 . 01 , ns , not significant .

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Figure 5Representative images (A and E), histological sections of dorsal back (B and F) and clinical scores (C and G) and quantitative PCR analysis of mRNA levels in dorsal skin (D and H) from wild-type (n=6) and M-Shp2-/- mice or DC-Shp2-/- mice (n=6) treated with IMQ for 4 days. Scale bar: 100 μm. Left: H&E (hematoxylin and eosin) staining data; right: statistical data (mean ± SEM). Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values were calculated by Tukey multiple comparison test (B, D, F, H) or Bonferroni multiple comparison test (C, G). *P<0.05, **P<0.01, ns, not significant.


Figure 6A Heat map showing mRNA expression in peritoneal macrophages derived from wild - type and M - Shp2 - / - mice after IMQ treatment based on RNA sequencing ( n = 3 / group ) . Colors represent high ( red ) and low ( blue ) intensity . Genes labeled in green indicate they are in the NF - κB signaling . D BMDMs and PMs derived from wild - type and M - Shp2 - / - mice were stimulated by IMQ ( 10 µg / ml ) for indicated times . Whole cell lysates were subjected to Western blotting . E Representative p - p65 staining of skin sections from mouse . Scale bar : 100 μm .

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Figure 6A Heat map showing mRNA expression in peritoneal macrophages derived from wild-type and M-Shp2-/- mice after IMQ treatment based on RNA sequencing (n=3/group). Colors represent high (red) and low (blue) intensity. Genes labeled in green indicate they are in the NF-κB signaling.D BMDMs and PMs derived from wild-type and M-Shp2-/- mice were stimulated by IMQ (10 µg/ml) for indicated times. Whole cell lysates were subjected to Western blotting.E Representative p-p65 staining of skin sections from mouse. Scale bar: 100 μm.


Figure 7E Immunoblotting of HEK293T cells co - transfected for 48 h with GFP - TLR7 , plus HA - SHP2 , HA - SHP2 mutant vectors , followed by immunoprecipitation with anti - GFP beads . F PMA - differentiated THP - 1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 ( 10 µg / ml ) . Subcellular fractionation was performed , and cytoplasmic and cell membrane proteins were probed by respective antibodies . G , H Immunoblot analysis of TLR7 and SHP2 in the isolated ER , Golgi and endosome from PMA - differentiated THP - 1 cells with either SHP2 overexpressed ( G ) and SHP2 deficiency ( H ) . I Skin sections from psoriatic patients and healthy controls were immune - stained for TLR7 together with a marker of the early endosome ( EEA1 ) or late endosome / lysosome ( LAMP1 ) prior to analysis by confocal microscopy , showing TLR7 localization in different organelles ( arrows ) . Scale bar : 50 μm .

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Figure 7E Immunoblotting of HEK293T cells co-transfected for 48 h with GFP-TLR7, plus HA-SHP2, HA-SHP2 mutant vectors, followed by immunoprecipitation with anti-GFP beads.F PMA-differentiated THP-1 cells were infected with vector or SHP2 lentivirus and then treated with or without R848 (10 µg/ml). Subcellular fractionation was performed, and cytoplasmic and cell membrane proteins were probed by respective antibodies.G, H Immunoblot analysis of TLR7 and SHP2 in the isolated ER, Golgi and endosome from PMA-differentiated THP-1 cells with either SHP2 overexpressed (G) and SHP2 deficiency(H).I Skin sections from psoriatic patients and healthy controls were immune-stained for TLR7 together with a marker of the early endosome (EEA1) or late endosome/lysosome (LAMP1) prior to analysis by confocal microscopy, showing TLR7 localization in different organelles (arrows). Scale bar: 50 μm.


Figure 8B , Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP - TLR7 or GFP - TLR7 mutant vectors . G HEK293T cells transfected with NF - κB - luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ ( 10 µg / ml ) for 24 h were harvested for luciferase assay . Data information : Data are represented mean ± SEM . P values are determined by two - tailed unpaired Student ' s t - test * * P < 0 . 01 , ns , not significant . I Representative p - TLR7 Y1024 staining of skin sections from human . Scale bar : 100 μm .

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Figure 8B, Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP-TLR7 or GFP-TLR7 mutant vectors.G HEK293T cells transfected with NF-κB-luciferase reporter and indicated TLR7 mutant vectors and then treated with IMQ (10 µg/ml) for 24 h were harvested for luciferase assay. Data information: Data are represented mean ± SEM. P values are determined by two-tailed unpaired Student's t-test **P<0.01, ns, not significant.I Representative p-TLR7 Y1024 staining of skin sections from human. Scale bar: 100 μm.


Figure 9Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . A Phenotypic presentation ( top ) and H & E staining ( bottom ) of dorsal skin . Scale bar : 100 μm . Left : H & E staining data ; right : statistical data ( mean ± SEM ) . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . D Quantitative PCR analysis of mRNA encoding IL - 23 / IL - 17A axis cytokines and other psoriasis - related cytokines in the dorsal skin . Results were normalized to Gapdh expression . Data information : Data are represented mean ± SEM . P values are determined by two - tailed Mann - Whitney U test * P < 0 . 05 , * * P < 0 . 01 . Tlr7wt / wt female mice ( n = 6 ) and Tlr7 - Y1025D mutant Tlr7ki / wt female mice ( n = 6 ) and Tlr7ki / ki female mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . E Representative p - p65 staining of the dorsal skin . Scale bars : 100 μm .

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Figure 9Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. A Phenotypic presentation (top) and H&E staining (bottom) of dorsal skin. Scale bar: 100 μm. Left: H&E staining data; right: statistical data (mean ± SEM). Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. D Quantitative PCR analysis of mRNA encoding IL-23/IL-17A axis cytokines and other psoriasis-related cytokines in the dorsal skin. Results were normalized to Gapdh expression. Data information: Data are represented mean ± SEM. P values are determined by two-tailed Mann-Whitney U test *P<0.05, **P<0.01.Tlr7wt/wt female mice (n=6) and Tlr7-Y1025D mutant Tlr7ki/wt female mice (n=6) and Tlr7ki/ki female mice (n=5) were treated with indicated dose of IMQ for 4 days. E Representative p-p65 staining of the dorsal skin. Scale bars: 100 μm.


Figure 1A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21 % O2 vs . 85 % O2 , at post - natal day ( P ) 5 and P14 . B Quantitative RT - PCR detection of microRNA - 34a / b / c - 5p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization . C Quantitative RT - PCR detection of microRNA - 34a / b / c - 3p family members in the lung over the course of normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization .

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Figure 1A Microarray analysis of microRNA expression changes in newborn mouse lungs exposed to 21% O2 vs. 85% O2, at post-natal day (P)5 and P14.B Quantitative RT-PCR detection of microRNA-34a/b/c-5p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization. C Quantitative RT-PCR detection of microRNA-34a/b/c-3p family members in the lung over the course of normal (21% O2) and aberrant (85% O2) alveolarization.


Figure 2A Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34a - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . B Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization . C Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34a - / - mice ( 34a - / - ) during normal and aberrant alveolarization . D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) and miR - 34bc - / - mice during normal and aberrant alveolarization ( scale bar , 50 µm ) . E Quantification of total number of alveoli by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization . F Quantification of mean septal thickness by design - based stereology in wild - type ( WT ) and miR ‑ 34bc - / - mice ( 34bc - / - ) during normal and aberrant alveolarization . G Localization of miR - 34a expression by β - galactosidase activity staining in the developing lungs of P14 miR - 34a : : lacZ + / + mice that were undergoing normal or aberrant alveolarization ( scale bar , 50 µm ; larger and some additional images are presented in Appendix Fig S2 ) .

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Figure 2A Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34a-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). B Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. C Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34a-/- mice (34a-/-) during normal and aberrant alveolarization. D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type (WT) and miR-34bc-/- mice during normal and aberrant alveolarization (scale bar, 50 µm). E Quantification of total number of alveoli by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. F Quantification of mean septal thickness by design-based stereology in wild-type (WT) and miR‑34bc-/- mice (34bc-/-) during normal and aberrant alveolarization. G Localization of miR-34a expression by β-galactosidase activity staining in the developing lungs of P14 miR-34a::lacZ+/+ mice that were undergoing normal or aberrant alveolarization (scale bar, 50 µm; larger and some additional images are presented in Appendix Fig S2).


Figure 3B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA ( SCR ) or a miR - 34a ( MIM34a ) mimic ( n = 3 separate cell cultures for each group ) . C Quantitative RT - PCR detection of miR - 34a / b / c - 5p levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 ( n = 3 separate cell cultures for each group ) . D Immunoblot detection of PDGFRα levels in the lungs of mouse pups ( n = 6 animals for each group ) at post - natal day ( P ) 5 , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization . E Immunoblot detection of PDGFRα levels in MLg cells in vitro , maintained under 21 % O2 or 85 % O2 , where cells had been transfected wither with a scrambled ( SCR ) antimiR , or an antimiR directed against miR - 34a ( A34a ) ( n = 3 separate cell cultures for each group ) . F Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of mouse pups ( n = 4 animals for each group ; data from an independent repetition are provided in Appendix Fig S5 ) at P5 , maintained under 21 % O2 or 85 % O2 from birth . H Quantitative RT - PCR detection of miR - 34a - 5p levels in PDGFRα + cells , sorted by FACS from the lungs of either wild - type ( 34awt / wt ) mouse pups , or mouse pups in which miR - 34a expression in Pdgfra ‑ expressing cells ( 34ai ∆ PC / i ∆ PC ) at P5 ( n = 4 animals for each group ) . I Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 during aberrant ( 85 % O2 ) alveolarization , compared with 34ai ∆ PC / i ∆ PC during normal ( 21 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of observations made in four other experiments . J Quantification of total number of alveoli by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) . K Quantification of mean septal thickness by design - based stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

Figure 3B Immunoblot detection of PDGFRα levels in MLg cells after treatment with scrambled microRNA (SCR) or a miR-34a (MIM34a) mimic (n = 3 separate cell cultures for each group).C Quantitative RT-PCR detection of miR-34a/b/c-5p levels in MLg cells in vitro, maintained under 21% O2 or 85% O2 (n = 3 separate cell cultures for each group).D Immunoblot detection of PDGFRα levels in the lungs of mouse pups (n = 6 animals for each group) at post-natal day (P)5, during normal (21% O2) and aberrant (85% O2) alveolarization.E Immunoblot detection of PDGFRα levels in MLg cells in vitro, maintained under 21% O2 or 85% O2, where cells had been transfected wither with a scrambled (SCR) antimiR, or an antimiR directed against miR-34a (A34a) (n = 3 separate cell cultures for each group).F Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of mouse pups (n = 4 animals for each group; data from an independent repetition are provided in Appendix Fig S5) at P5, maintained under 21% O2 or 85% O2 from birth.H Quantitative RT-PCR detection of miR-34a-5p levels in PDGFRα+ cells, sorted by FACS from the lungs of either wild-type (34awt/wt) mouse pups, or mouse pups in which miR-34a expression in Pdgfra‑expressing cells (34ai∆PC/i∆PC) at P5 (n = 4 animals for each group).I Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14 during aberrant (85% O2) alveolarization, compared with 34ai∆PC/i∆PC during normal (21% O2) alveolarization (scale bar, 50 µm). Data are representative of observations made in four other experiments. J Quantification of total number of alveoli by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group). K Quantification of mean septal thickness by design-based stereology in 34awt/wt or 34ai∆PC/i∆PC mouse pups at P14, during normal and aberrant alveolarization (n = 5 animals for each group).


Figure 4B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and c - Kit as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and c - Kit were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments . C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR ( SCR ) and miR - 34a ( MIM34a ) mimics , and probing for PDGFRα and SIRT1 as TSB ‑ dependent and TSB ‑ independent target readouts , respectively . Protein loading equivalence was controlled by βactin levels . Note : PDGFRα , βactin , and SIRT1 were all probed on the same membrane ; hence a single βactin immunoblot is presented . Data are representative of three experiments . D Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled target site blocker ( SCR ) or a cocktail of both target site blockers ( TSB1 and TSB2 ) during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . E Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) . F Quantification of mean septal thickness by design - based stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) . G Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) . H Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or the TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 animals for each group ) .

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

Figure 4B Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and c-Kit as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and c-Kit were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.C Evaluation of the specificity of TSB1 and TSB2 in MLg cells using scrambled miR (SCR) and miR-34a (MIM34a) mimics, and probing for PDGFRα and SIRT1 as TSB‑dependent and TSB‑independent target readouts, respectively. Protein loading equivalence was controlled by βactin levels. Note: PDGFRα, βactin, and SIRT1 were all probed on the same membrane; hence a single βactin immunoblot is presented. Data are representative of three experiments.D Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled target site blocker (SCR) or a cocktail of both target site blockers (TSB1 and TSB2) during normal (21% O2) and aberrant (85% O2) alveolarization (scale bar, 50 µm). Data are representative of three or more experiments. E Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). F Quantification of mean septal thickness by design-based stereology in wild-type mouse pups at P14, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group). G Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).H Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with scrambled target site blocker (SCR) or the TSB1,2 cocktail during aberrant alveolarization (n = 5 animals for each group).


Figure 5B Quantitative RT - PCR detection of miR - 34a - 5p levels in wild - type mouse pups at post - natal day ( P ) 14 that had been treated either with a scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal ( 21 % O2 ) and aberrant ( 85 % O2 ) alveolarization ( n = 4 animals for each group ) . C Qualitative analysis of lung structure in Richardson - stained plastic - embedded lung sections from wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . D Quantification of total number of alveoli by design - based stereology in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant lung alveolarization ( n = 5 animals for each group ) . E Quantification of mean septal thickness by design - based stereology in in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) . F Quantitative analysis of PDGFRα + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) . G Quantitative analysis of PDGFRα + / αSMA + cells by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal and aberrant alveolarization ( n = 5 animals for each group ) .

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

Figure 5B Quantitative RT-PCR detection of miR-34a-5p levels in wild-type mouse pups at post-natal day (P)14 that had been treated either with a scrambled antimiR (S), or antimiR-34a (A34a), during normal (21% O2) and aberrant (85% O2) alveolarization (n = 4 animals for each group).C Qualitative analysis of lung structure in Richardson-stained plastic-embedded lung sections from wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (scale bar, 50 µm). Data are representative of three or more experiments.D Quantification of total number of alveoli by design-based stereology in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant lung alveolarization (n = 5 animals for each group).E Quantification of mean septal thickness by design-based stereology in in wild-type mouse pups at post-natal day (P)14, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).F Quantitative analysis of PDGFRα+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).G Quantitative analysis of PDGFRα+/αSMA+ cells by flow cytometry, in lungs from wild-type mouse pups at P5, treated with either scrambled antimiR (S), or antimiR-34a (A34a), during normal and aberrant alveolarization (n = 5 animals for each group).


Figure 6A Mice expressing nuclear ‑ localized GFP under the control of the Pdgfra promoter were maintained under normoxic ( 21 % O2 ) or hyperoxic ( 85 % O2 ) conditions , and lungs were harvested , processed and immunostained for Ki67 to determine proliferation status . DAPI staining revealed nuclei of all cells present in the section . Low - magnification images from individual channels are presented to the right of the merged ( large ) image first row of images . The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image , as well as visualization of a single Ki67 + , GFP + cell ( white arrowhead ) in all three channels separately . B The number of PDGFRα + cells in four microscopic fields was assessed for co - staining with an anti - Ki67 antibody to reveal proliferating cells . P values were calculated by unpaired Student ' s t ‑ test ( n = 4 fields for each group , trends are representative of those observed in two other experiments ) . C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild - type mice that were treated with an isotype - matched control IgG used for the Ki67 staining experiments . Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining . Scale bar : 100 μm . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole ; GFP , green fluorescent protein .

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

Figure 6A Mice expressing nuclear‑localized GFP under the control of the Pdgfra promoter were maintained under normoxic (21% O2) or hyperoxic (85% O2) conditions, and lungs were harvested, processed and immunostained for Ki67 to determine proliferation status. DAPI staining revealed nuclei of all cells present in the section. Low-magnification images from individual channels are presented to the right of the merged (large) image first row of images. The area demarcated by the white box in the merge image of the first row is magnified in the second and third rows to allow for visualization of greater magnification of the demarcated region of the merged image, as well as visualization of a single Ki67+,GFP+ cell (white arrowhead) in all three channels separately.B The number of PDGFRα+ cells in four microscopic fields was assessed for co-staining with an anti-Ki67 antibody to reveal proliferating cells. P values were calculated by unpaired Student's t‑test (n=4 fields for each group, trends are representative of those observed in two other experiments).C The Ki67 staining and GFP fluorescence was controlled for by examining lungs from wild-type mice that were treated with an isotype-matched control IgG used for the Ki67 staining experiments. Sections were examined for GFP fluorescence as well as in the red channel used to detect the Ki67 staining. Scale bar: 100 μm. DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein.


Figure 7The impact of administration of scrambled antimiR ( S ) or an antimiR directed against miR - 34a ( A34a ) on the abundance of type I alveolar epithelial cells ( marked by aquaporin 5 , Aqp5 ) and type II alveolar epithelial cells ( marked by pro - surfactant protein C , Sftpc ) was assessed in 3 - µm sections of paraffin ‑ embedded lung tissue from P5 mice undergoing normal ( 21 % O2 ) or aberrant ( 85 % O2 ) lung alveolarization . DAPI , 4 ′ , 6 - diamidino - 2 - phenylindole . In the DAPI images , white lines delineate tissue from airspaces , and in the 85 % O2 groups demarcate septa . Antibody specificity was validated by rabbit IgG isotype control primary antibodies . The control experiments for the Aqp5 and Sftpc staining runs are illustrated here . Scale bars , 50 µm .

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

Figure 7The impact of administration of scrambled antimiR (S) or an antimiR directed against miR-34a (A34a) on the abundance of type I alveolar epithelial cells (marked by aquaporin 5, Aqp5) and type II alveolar epithelial cells (marked by pro-surfactant protein C, Sftpc) was assessed in 3-µm sections of paraffin‑embedded lung tissue from P5 mice undergoing normal (21% O2) or aberrant (85% O2) lung alveolarization. DAPI, 4′,6-diamidino-2-phenylindole. In the DAPI images, white lines delineate tissue from airspaces, and in the 85% O2 groups demarcate septa. Antibody specificity was validated by rabbit IgG isotype control primary antibodies. The control experiments for the Aqp5 and Sftpc staining runs are illustrated here. Scale bars, 50 µm.


Figure 1D ) HEK293 cells were genetically engineered to knockout RIG - I , MDA5 , or both , and subsequently subjected to retroviral transduction to stably express FLAG - LGP2 or an empty vector ( EV ) . Correct gene editing and intact type I IFN responsiveness was validated by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . * , non - specific band . E ) Cells generated in ( D ) were transfected with siCtrl or siADAR1 . The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post - transfection as in ( B ) . Data are means ± s . d . from a representative of four biological replicate experiments . F ) Cells generated in ( D ) were transfected with siCtrl ( siC ) or siADAR1 ( siA ) . Protein lysates were prepared 78h post - transfection , followed by SDS - PAGE and immunoblotting using the indicated antibodies ( n = 2 ) . G ) HEK293 WT cells and FLAG - LGP2 - expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence . Cells were fixed , permeabilized and stained 72h post - transfection with anti - FLAG ( red ) and anti - IRF3 ( green ) antibodies . Nuclei were stained with DAPI ( blue ) . Scale bar is 50 μm . Total nuclei ( > 450 nuclei per experimental condition ) and IRF3 - positive nuclei were counted using semi - automated software analysis and plotted as percentage IRF3 - positive nuclei of total nuclei per field of view ( a representative of three biological replicate experiments is quantified ) . The boxplot indicates the interquartile range as a box , the median as a central line , and the whiskers extend from the minimum to the maximum value . Statistical analyses were performed using unpaired two - tailed Mann - Whitney U tests . ns , not significant ; * * * * , p < 0 . 0001 .

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

Figure 1D) HEK293 cells were genetically engineered to knockout RIG-I, MDA5, or both, and subsequently subjected to retroviral transduction to stably express FLAG-LGP2 or an empty vector (EV). Correct gene editing and intact type I IFN responsiveness was validated by SDS-PAGE and immunoblotting using the indicated antibodies (n=2). *, non-specific band.E) Cells generated in (D) were transfected with siCtrl or siADAR1. The type I IFN response and ADAR1 knockdown efficiency was monitored 78h post-transfection as in (B). Data are means ± s.d. from a representative of four biological replicate experiments.F) Cells generated in (D) were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 78h post-transfection, followed by SDS-PAGE and immunoblotting using the indicated antibodies (n=2).G) HEK293 WT cells and FLAG-LGP2-expressing HEK293 cells were transfected with siCtrl or siADAR1 and subsequently plated on coverslips for immunofluorescence. Cells were fixed, permeabilized and stained 72h post-transfection with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>450 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of three biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analyses were performed using unpaired two-tailed Mann-Whitney U tests. ns, not significant; ****, p<0.0001.


Figure 2A ) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR / Cas9 . Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness , respectively . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 3 ) . B ) ADAR1 - knockout HEK293 cells ( clone 1 ) were co - transfected with an empty vector ( EV ) or a FLAG - LGP2 - encoding vector ( LGP2 ) combined with a vector encoding GFP - tagged ADAR1 p110 or p150 . Cells were harvested 72h post - transfection and the type I IFN response was monitored by RT - qPCR analysis of IFN - β and IFIT1 expression , normalized to ACTB . Data are means ± s . d . from a representative of four biological replicate experiments . C ) ADAR1 - knockout cells ( clone 1 ) were transfected as in ( B ) . Protein lysates were analyzed by SDS - PAGE followed by immunoblotting using the indicated antibodies ( n = 4 ) .

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

Figure 2A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).