( A ) Lung cancer PC - 9 , prostate cancer 22RV1 , bladder cancer UMUC3 , breast cancer MCF - 7 , colonic adenoma - derived non - cancerous AA / C1 ( aka C1 ) AA / C1 / SB ( aka SB ) , or cancerous AA / C1 / SB / 10C ( aka 10C ) were incubated with wild - type F . nucleatum 12230 ( Fn ) , fadA - deletion mutant US1 ( US1 ) , or E . coli DH5α ( E . coli ) at multiplicity of infection ( MOI ) of 1 , 000 : 1 . numbers are mean values ± . experiment was performed in triplicates repeated three times . * * p < 0 . 01 , * * * p < 0 . 001 , compared to untreated controls ; # p < 0 . 05 , # # # p < 0 . 001 , compared to US1 - treated ( two - way ANOVA ) .

O O O B-DISEASE I-DISEASE B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-CELL_LINE O B-DISEASE I-DISEASE B-CELL_LINE O B-DISEASE I-DISEASE B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-DISEASE I-DISEASE O O O O B-DISEASE O O O O O O O O O O O O O O O O O O B-DISEASE O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O B-GENEPROD O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lung cancer cells PC-9, prostate cancer cells 22RV1, bladder cancer cells UMUC3, breast cancer cells MCF-7, colonic adenoma-derived non-cancerous cells AA/C1 (aka C1) and AA/C1/SB (aka SB), or cancerous cells AA/C1/SB/10C (aka 10C) were incubated with wild-type F. nucleatum 12230 (Fn), the fadA-deletion mutant US1 (US1), or E. coli DH5α (E. coli) at multiplicity of infection (MOI) of 1,000:1. Cells numbers are mean values ± SEM. The experiment was performed in triplicates and repeated three times. **p<0.01, ***p<0.001, compared to untreated controls; #p<0.05, ### p<0.001, compared to US1-treated cells (two-way ANOVA).


( B ) Attachment ( left panel ) invasion ( right panel ) of wild - type F . nucleatum 12230 ( Fn ) to non - cancerous SB , either untreated or transfected with antisense or sense ANXA1 , to cancerous 10C , either untreated or transfected with control or ANXA1 - or CDH1 - specific siRNA or both ( MOI 50 : 1 ) . F . nucleatum attachment invasion to untreated SB were designated as 100 % , respectively ; other values were expressed as relative to those obtained with untreated SB . Data are mean values ± . experiment was performed in triplicates repeated four times . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ( one - way ANOVA ) .

O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O B-DISEASE O O O O O O O O O O B-GENEPROD O O B-DISEASE O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Attachment (left panel) and invasion (right panel) of wild-type F. nucleatum 12230 (Fn) to the non-cancerous SB cells, either untreated or transfected with antisense or sense ANXA1, and to the cancerous 10C cells, either untreated or transfected with control or ANXA1- or CDH1-specific siRNA or both (MOI 50:1). F. nucleatum attachment and invasion to the untreated SB cells were designated as 100%, respectively; all other values were expressed as relative to those obtained with untreated SB. Data are mean values ± SEM. The experiment was performed in triplicates and repeated four times. *p<0.05, **p<0.01 and ***p<0.001 (one-way ANOVA).


( C ) Left panel : qPCR of Villin1 ( VIL1 ) mRNA in 10C treated with control siRNA or VIL1 - specific siRNA , demonstrating knocking down of Villin1 . Right panel : attachment of F . nucleatum 12230 to 10C treated with control siRNA or VIL1 - specific siRNA . Data are mean values ± SD . experiment was performed in triplicates repeated twice . * p < 0 . 05 ( student ' s t - ) .

O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Left panel: qPCR analysis of Villin1 (VIL1) mRNA levels in 10C cells treated with control siRNA or VIL1-specific siRNA, demonstrating knocking down of Villin1. Right panel: attachment of F. nucleatum 12230 to 10C cells treated with control siRNA or VIL1-specific siRNA. Data are mean values ± SD. The experiment was performed in triplicates and repeated twice. *p<0.05 (student's t-test).


Relationship between ANXA1 mRNA expression disease - free survival ( DFS ) in colon cancer . relationship between ANXA1 mRNA expression DFS was investigated in a database of 466 colon carcinomas , assembled by pooling four independent gene - expression array datasets from NCBI - GEO online repository ( GSE14333 , GSE17538 , GSE31595 , GSE37892 ) association between ANXA1 expression DFS was tested using Kaplan - Meier survival curves , after stratification in groups with high , medium low ANXA1 expression , using three different methods : ( A ) on median of ANXA1 mRNA expression ( high 50 % vs . low 50 % ) ; ( B ) on quartile distribution of ANXA1 mRNA expression ( high 25 % vs . middle 50 % vs . low 25 % ) ; ( C ) on ANXA1 mRNA expression thresholds calculated using StepMiner algorithm ( low vs . high ) Overall , high ANXA1 mRNA expression were associated with a statistically significant reduction in DFS ( p < 0 . 001 , log - rank ) , irrespective of method used for stratification . Differences in ANXA1 mRNA expression did not appear to correlate with differences in each ' s relative content of epithelial in analyzed biospecimens ( i . e . density ) as revealed by lack of visual correlations with epithelial marker Desmoplakin ( DSP ) .

O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-DISEASE I-DISEASE O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-DISEASE I-DISEASE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-DISEASE I-DISEASE O O O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O

Relationship between ANXA1 mRNA expression levels and disease-free survival (DFS) in colon cancer patients. The relationship between ANXA1 mRNA expression levels and DFS was investigated in a database of 466 primary colon carcinomas, assembled by pooling four independent gene-expression array datasets from the NCBI-GEO online repository (GSE14333, GSE17538, GSE31595, GSE37892) The association between ANXA1 expression levels and DFS was tested using Kaplan-Meier survival curves, after patient stratification in groups with high, medium and low ANXA1 expression, using three different methods: (A) based on the median of ANXA1 mRNA expression levels (high 50% vs. low 50%); (B) based on the quartile distribution of ANXA1 mRNA expression levels (high 25% vs. middle 50% vs. low 25%); and (C) based on ANXA1 mRNA expression thresholds calculated using the StepMiner algorithm (low vs. high) Overall, high ANXA1 mRNA expression levels were associated with a statistically significant reduction in DFS (p<0.001, log-rank test), irrespective of the method used for the stratification. Differences in ANXA1 mRNA expression levels did not appear to correlate with differences in each tumor's relative content of epithelial cells in the analyzed biospecimens (i.e. tumor cell density) as revealed by the lack of visual correlations with the epithelial cell marker Desmoplakin (DSP).


( A ) Real - time qPCR of ANXA1 expression using mRNA extracted from non - cancerous SB , cancerous 10C , human CRC lines HCT116 , DLD1 RKO , each grown to 50 % or 100 % confluency . results were normalized to ANXA1 mRNA in SB of 100 % confluency , which was designated as 1 . Data are mean values ± . experiment was performed in triplicates repeated twice . * * p < 0 . 01 * * * p < 0 . 001 ( student ' s t - ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O O B-DISEASE O O B-DISEASE O O B-ORGANISM B-DISEASE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of SB 10C grown to 20 % ( top panels ) or 100 % ( bottom panels ) confluency followed by immuno - of Annexin A1 ( green ) . nuclei were stained with DAPI ( blue ) . A series of 20 - 50 consecutive in z - axis were stacked together to generate 3 - D figure at 400x magnification . Annexin A1 is most abundantly expressed on 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 . experiment was repeated at least twice .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O 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-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 ) proliferation of adenoma - derived non - cancerous SB cancerous 10C human CRC lines HCT116 , DLD1 , SW480 , HT29 RKO either untreated ( black lines ) or following treatment with control siRNA ( gray lines ) or ANXA1 - specific siRNA ( green lines ) . Data are mean values ± . experiment was performed in triplicates repeated three times . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 , compared to untreated ( two - way ANOVA ) .

O O O B-EXP_ASSAY O B-DISEASE O O O O B-DISEASE O B-DISEASE O B-ORGANISM B-DISEASE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) proliferation of SB ( left panel ) RKO ( right panel ) transfected with ANXA1 ( red ) , as compared to control ( black lines ) . Data are mean values ± . experiment was performed in triplicates repeated three times . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 growth in nude mice following subcutaneous bilateral inoculation of HCT116 transfected with control siRNA or ANXA1 - specific siRNA . volumes were measured after 7 days post injection ( left panel , n = 4 ) . For each mouse , resulting from ANXA1 - specific siRNA treated was normalized to that from control siRNA - treated , which was designated 100 % . represents average . * p < 0 . 05 ( paired t - ) . pairs are shown on right panel : top , tumors arising from control siRNA treated ; bottom , tumors arising from ANXA1 - specific siRNA treated .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O 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-DISEASE O O O O O O O O B-DISEASE O O B-GENEPROD O O O O O

(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.


( A ) Real - time qPCR of ANXA1 mRNA in SB , 10C , HCT116 DLD1 incubated with wild - type F . nucleatum 12230 ( Fn ) or fadA - deletion mutant US1 ( US1 ) at MOI of 50 : 1 for indicated time periods . results were normalized to those obtained with untreated were mean of 3 independent experiments each performed in triplicates . * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD B-EXP_ASSAY O O O O O B-CELL_LINE I-CELL_LINE O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of Annexin A1 expression in 10C 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 . experiment was performed in triplicates repeated more than three times . * * p < 0 . 001 ( one - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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) 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 of Annexin A1 in 10C transfected with control siRNA ( dotted black ) or CDH1 - specific siRNA ( dotted red ) 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 . experiment was performed in triplicates repeated twice . * * * p < 0 . 001 ( two - way ANOVA ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 of SB 10C 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 E - cadherin blue . Imagines are 800x magnification . Note enhanced expression of Annexin A1 in 10C compared to SB its location on outer layer of mass . experiment was repeated three times . Scale bar , 250 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM O O O O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O

(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.


( A ) Western blotting of FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) β - catenin in DLD1 following incubation with FadAc for 15 or 120 min . C , untreated . β - actin was included as an internal control . experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_LINE O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

(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 lysates were incubated with FadAc for 15 or 120 min 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 ) β - catenin in eluates were detected by Western blot . C , untreated control . experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-CELL_LINE O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O

(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 lysates were incubated with FadAc for 15 or 120 min mixed with agarose beads conjugated with mouse anti - FadA monoclonal antibody ( α - FadA ) or control mouse IgG . FadA , E - cadherin ( CDH1 ) , Annexin A1 ( ANXA1 ) β - catenin in eluates were detected by Western blot . experiment was repeated three times .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_LINE O O O O B-SUBCELLULAR O O O O O O O B-SMALL_MOLECULE O O O B-ORGANISM O O B-GENEPROD O O O O O B-GENEPROD O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(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 of 10C either untreated ( top panel ) or following incubation with CFSE - labeled F . nucleatum 12230 ( red , bottom panel ) for 3 hrs then immunofluorescent - stained for Annexin A1 ( green ) E - cadherin ( blue ) . are 1200x magnification . A side view of enlarged image is shown on far right . Note enhanced expression of Annexin A1 in F . nucleatum - bound co - localization of Annexin A1 , E - cadherin F . nucleatum on membranes ( arrows ) . experiment was repeated more than three times . Scale bar , 500 nm .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-ORGANISM I-ORGANISM I-ORGANISM O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(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 of 10C following incubation with Alexa Fluor ™ 488 - conjugated BSA , mFadA , or FadAc ( red ) for 1 hr immuno - of Annexin A1 ( green ) E - cadherin ( blue ) . are 1200x magnification . Note enhanced expression of Annexin A1 its co - localization with E - cadherin in presence of FadAc ( arrows ) , but not with BSA mFadA . experiment was repeated twice . side views are shown to right bottom of each image . Scale bar , 500 nm .

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(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.


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

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Flow 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).


( 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 mouse colon are shown on right , pointed by blue arrows on right . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA ) .

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(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 in Apcmin / + mouse colonic tumors 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 ) .

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(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 ) ANXA1 mRNA ( y - axis ) in F . nucleatum - induced Apcmin / + mouse colonic tumors ( n = 34 ; Pearson ' s correlation ) . Each dot represents average of qPCR results performed in

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(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 paired normal adenocarcinoma tissues from human CRC ( n = 18 ) expressed as of fadA over total 16S rRNA genes determined by qPCR . Each symbol shows average of duplicate qPCR results from one . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - ) .

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(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 in paired normal adenocarcinoma tissues from CRC ( n = 18 ) . Each symbol shows average of duplicate qPCR results from one . Horizontal lines represent mean values . * * p < 0 . 01 ( paired t - ) .

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(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 ) ANXA1 mRNA ( y - axis ) in human colorectal adenocarcinoma tissues ( n = 18 ; Pearson ' s correlation ) . Each dot represents average of qPCR results performed in duplicates .

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(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 of paired normal carcinoma tissues from two colon cancer . frozen sections were incubated with rabbit anti - AnnexinA1 polyclonal antibodies 5G11 mouse anti - FadA monoclonal antibodies . slides were then stained with Alexa Fluor ® 680 - conjugated donkey anti - rabbit Alexa Fluor ® 555 - conjugated goat anti - mouse , washed , covered in mounting medium containing DAPI . scanning confocal microscopy mages were taken with a Nikon Ti Eclipse inverted microscope at 200x magnification for normal tissues 400x for carcinomas . Scale bar , 50 μm . Co - localization of FadA ( red ) Annexin A1 ( green ) was observed in carcinomas but not in paired normal tissues .

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(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.


( A ) Flow cytometry of β - catenin expression in 10C , HCT116 DLD1 following transfection with control siRNA ( clear bars ) or ANXA1 - specific siRNA ( solid bars ) incubation with F . nucleatum 12230 at MOI of ~ 20 : 1 for indicated time periods . geometric means of treated with control siRNA at time 0 was designated as 1 . Data are mean values ± SD . experiment was performed in duplicates or triplicates repeated 1 - 3 times . * * p < 0 . 01 , * * * p < 0 . 001 ( two - way ANOVA ) .

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(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 - of β - catenin in 10C following transfection with control siRNA or ANXA1 - specific siRNA incubation with F . nucleatum 12230 ( Fn ) at MOI of ~ 100 : 1 for 2 hrs . β - catenin was stained with Alexa Fluor ® 680 ( red ) nuclei with DAPI ( blue ) . were captured with confocal microscope at 800x magnification . - , no added . Note increased expression of β - catenin its nucleus translocation in response to F . nucleatum in control siRNA - treated , but not in ANXA1 siRNA - treated . experiment was repeated twice . Scale bar , 200 nm .

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(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 of CCND1 expression using mRNA extracted from cancerous 10C , human CRC HCT116 RKO , each grown to 50 % or 100 % confluency . Data are mean values ± . experiment was performed in triplicates repeated twice . * p < 0 . 05 , * * * p < 0 . 001 ( student ' s t - ) .

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(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 of Cyclin D1 , Annexin A1 , β - actin in RKO transfected with control vector or ANXA1 . Induction of Cyclin D1 was observed in response to increased Annexin A1 . experiment was repeated twice .

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(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.


( A ) Tru - Seq of E12 . 5 midbrain floor - plate ( mFP ) , midbrain roof - plate ( mRP ) , anterior ( A , adjacent anterior FP ) posterior ( P , adjacent posterior FP ) . Pbx1 is enriched in midbrain FP , together with Th Pitx3 . Lower of Pbx3 are also expressed in mFP . Gdf7 Wnt1 are restricted to mRP at E12 . 5 .

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(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 intermediate zone ( IZ ) marginal zones ( MZ ) , but not ventricular zone ( VZ ) , of mFP at E12 . 5 , as detected by in situ hybridization .

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(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 ventro - lateral part of LMX1a + domain at E10 , preceding birth of first ( TH + ) mDA neurons at E10 . 5 . At E12 . 5 , PBX1 is present in mDA neurons , but not PBX1 + are TH + . boxes indicate showed in higher magnification ( right ) .

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(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 defines a subpopulation of NURR1 + neuroblasts labels NURR1 + TH + mDA neurons .

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(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 is also detected in a subpopulation of NURR1 + PITX3 - postmitotic neuroblasts at E12 . 5 . Higher magnification revealed three different populations of postmitotic : neuroblasts ( NURR1 + PBX1A - PITX3 - , green ) , secondary neuroblasts ( NURR1 + PBX1A + PITX3 - , yellow / orange ) tertiary neuroblasts / mDA neurons ( NURR1 + PBX1A + PITX3 + , ) . , Dapi ( 4 ′ , 6 - diamidino - 2 - phenylindole , blue ) . scale bars , 20µm .

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(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.


( A ) PBX1A is isoform detected in TH + NURR1 + mDA neurons at E12 . 5 .

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(A) PBX1A is the isoform detected in the TH+NURR1+mDA neurons at E12.5.


( C ) Immunofluorescence microscopy of 7 - week old human VM tissue showing that mDAn are positive for PBX1 , suggesting a conserved role for this factor in development of these neurons in mice humans .

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(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 substantia nigra ( SN ) ventral tegmental ( VTA ) .

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(D) PBX1 is also present in TH+ adult mDA neurons of the substantia nigra (SN) and ventral tegmental area (VTA).


( A ) To left , representative immunofluorescence microscopy / confocal showing that number of TH + is reduced in Pbx1 - / - . Deletion of Pbx1 decreases proportion of NURR1 + neuroblasts that become TH + neurons at E12 . 5 , indicating that Pbx1 is required for proper maturation of NURR1 + neuroblasts . To right , of these experiments .

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(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 rostro - caudal of VM in Pbx1 - / - mice at E12 . 5 , indicating a repressive function of PBX1 on Pbx3 expression .

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(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 showing that conditional deletion of Pbx1 Pbx3 ( cKO , Pbx1flox / flox ; Pbx3 - / - ; Th - IRES - Cre - ERT ) reduces number of PITX3 + in rostral VM , but not of NURR1 + , when compared to double heterozygous at E14 . 5 ( cHET , Pbx1flox / + ; Pbx3 - / + ; Th - IRES - Cre - ERT ) . boxes are magnified to right .

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(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 + NURR1 + is detected from anterior to posterior in cKO , but not in cHET . Pyknotic NURR1 + nuclei are observed in cKO ( arrowheads ) . boxes are magnified to right .

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(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 ) total number of TH + mDA neurons is dramatically reduced there is an increase in aCASP3 + in cKO compared to cHET mice at E18 . 5 .

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(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.


( B ) Representative immunofluorescence microscopy / confocalimages showing that PBX1 - overexpression increases number of TH + neurons after 8 days of differentiation of hNES ( AF22 derived from induced pluripotent ) . ( C ) While number of ( Dapi + ) in both conditions did not change , number of TH + increased 2 . 3 fold in PBX1 - overexpressing .

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(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 + derived from PBX1 - overexpressing hNES were also positive for mDAn markers such as LMX1A PITX3 . scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates per condition p - value ( T - ) are indicated in graphs .

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(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.


( B ) Schematic of 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 .

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(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 29 most regulated genes ( t - q - value < 0 . 05 ) identified by Gene Set Enrichment ( GSEA ) ranked by p - value . RPKM stands for Reads Per Kilobase of transcript per Million mapped reads . GSEA was performed on genes identified by - Seq as differentially expressed in VM at E12 . 5 compared to adjacent brain regions identified by PBX1Chip - Seq in proximity to transcription starting sites .

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(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 GSEA curve showing a high correlation between cistrome transcriptome ( p - value < 0 . 001 , q - value < 0 . 005 ) .

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(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 TSS of Pitx3 ( Pitx3 - Gbf1 ) , Onecut2and Nfe2l1 genes . No co - immunoprecipitation was detected for unrelated gene , Lrp1b ( Low Density Lipoprotein Receptor - Related 1B ) .

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(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 ) regulation of Pitx3 Onecut2 by PBX1 was confirmed in Pbx1 - / - ; Pbx3 + / - mutant at E12 . 5 , compared to wild - type ( Pbx1 + / + ) . ( F ) Representative immunofluorescence microscopy / confocal showing that decreased of PITX3 number of PITX3 + in E12 . 5 Pbx1 - / - ; Pbx3 + / - . This reduction is more evident in basal plate ( BP ) in midline ( M ) , as shown at higher magnification ( right ) . ( G ) Double ONECUT2 + TH + were detected only in lateral part of mFP in E12 . 5 Pbx1 - / - ; Pbx3 + / - . Selected areas are magnified to right . A scheme ( right ) shows distribution of in Pbx1 + / + Pbx1 - / - ; Pbx3 + / - . scale bars , 40µm . Error bars represent mean ± standard deviation . Biological replicates ( dots ) per condition p - value ( T - ) are indicated in each graph .

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(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.


( A ) Representative immunofluorescence microscopy / confocal showing that NFE2L1 is detected in TH + mDAn neuroblasts at E12 . 5 .

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(A) Representative immunofluorescence microscopy/confocal images showing that NFE2L1 is detected in TH+mDAn and neuroblasts at E12.5.


( B ) expression of NFE2L1 in TH + neurons is dramatically reduced in Pbx1 - / - ; Pbx3 + / - compared to Pbx1 + / + at E12 .

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(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 of aCASP3 in H2O2 - treated ( 100µm for 12 hours ) TH + neurons derived from hNES differentiated for 8 days ( ) . arrows indicate double TH + aCASP3 + neurons .

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(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 from oxidative stress induced by H2O2 ( 100µm for 12 hours ) , resulting in reduced of aCASP3 in TH + neurons ( ) .

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(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 ( - blue / black , indicated by arrowheads ) in nuclei of neuromelanin + ( NM + , brown ) of SN . In contrast , PBX1 was severely reduced in PD . ( F ) Graphical representation of PBX1 in SN of NM + neurons in controls PD analyzed . Each dot in graph represents level of PBX1 in nuclei of one single NM + neuron .

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(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 PBX1 direct target , NFE2L1 , was also detected as very well defined punctuate structures in NM + of SN in control samples ( arrowheads ) , but were dramatically reduced in PD . Yellow asterisks indicate nucleoli . ( H ) of NM + neurons showing NFE2L1 in SN of control donors PD . ( I ) Graphical representation of controls PD analyzed , showing that NM + neurons with NFE2L1 + nuclei in SN of PD are less than 10 % or are totally absent .

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(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.


A Expression of PTPN11 ( gene encoding SHP2 ) in skin lesions in psoriatic compared with skin from healthy donors on microarray data ( No . GSE14905 ) . B Expression of ptpn11 in human PBMCs of psoriatic ( n = 14 ) normal controls ( n = 16 ) . Data information : Data are represented mean ± . P values are determined by two - Mann - Whitney U * P < 0 . 05 , * * P < 0 . 01 .

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A 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 in skin sections of psoriatic ( n = 13 ) normal controls ( n = 5 ) . Scale bars : 200 μm .

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C Representative SHP2 staining in skin sections of psoriatic patients (n=13) and normal controls (n=5). Scale bars: 200 μm.


D Western blot of PBMCs lysates derived from psoriatic normal controls .

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D Western blot analysis of PBMCs lysates derived from psoriatic patients and normal controls.


E catalytic activity of SHP2 was measured in human PBMCs lysates derived from psoriatic ( n = 25 ) normal controls ( n = 25 ) . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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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 of skin sections of psoriatic normal controls . Scale bars : 200 μm .

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F Representative p-ERK staining of skin sections of psoriatic patients and normal controls. Scale bars: 200 μm.


G Quantitative PCR of Ptpn11 mRNA in 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 ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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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 .

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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 of mRNA in human PBMCs derived from psoriatic ( n = 20 ) normal healthy controls ( n = 20 ) . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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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.


C57BL / 6 mice ( n = 6 / group ) were treated with indicated dose of SHP099 or vehicle for 4 days . A Phenotypic presentation ( top ) , H & E ( middle ) statistical data ( bottom ) ( mean ± ) of dorsal skin . Scale bar : 100 μm . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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C57BL/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 of mRNA encoding IL - 23 / IL - 17A axis cytokines in 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 ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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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 of of cytokines in mouse serum . Quantitative PCR of mRNA encoding psoriasis - related cytokines in 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 ± . P values are determined by two - unpaired Student ' s t - * P < 0 . 05 , * * P < 0 . 01 .

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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.


Violin plot showed relative expression of 47 inflammation - associated genes on sample groups clusters .

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


D Gene set enrichment ( GSEA ) of NF - κB signaling pathway .

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D 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 . Examples of DEGs were labeled .

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E Volcano plots of DEGs that were upregulated (red) or downregulated (blue) in myeloid cells. Examples of DEGs were labeled.


G Expression patterns of top ten hub genes in ( F ) . ( top ) displays average expression in three groups in different myeloid subsets . Boxplots ( bottom ) show corresponding expression distribution .

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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.


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

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Representative 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.


A Heat map showing mRNA expression in peritoneal macrophages derived from wild - type M - Shp2 - / - mice after IMQ treatment on sequencing ( n = 3 / group ) . Colors represent high ( red ) low ( blue ) . Genes labeled in green indicate they are in NF - κB signaling .

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A 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 PMs derived from wild - type M - Shp2 - / - mice were stimulated by IMQ ( 10 µg / ml ) for indicated times . Whole lysates were subjected to Western blotting .

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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 of skin sections from mouse . Scale bar : 100 μm .

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E Representative p-p65 staining of skin sections from mouse. Scale bar: 100 μm.


E Immunoblotting of HEK293T co - transfected for 48 h with GFP - TLR7 , plus HA - SHP2 , HA - SHP2 mutant vectors , followed by immunoprecipitation with anti - GFP beads .

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E 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 were infected with vector or SHP2 lentivirus then treated with or without R848 ( 10 µg / ml ) . Subcellular fractionation was performed , were probed by respective antibodies .

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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 of TLR7 SHP2 in isolated ER , Golgi endosome from PMA - differentiated THP - 1 with either SHP2 overexpressed ( G ) SHP2 deficiency ( H ) .

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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 healthy controls were immune - stained for TLR7 together with a marker of early endosome ( EEA1 ) or late endosome / lysosome ( LAMP1 ) prior to by confocal microscopy , showing TLR7 localization in different organelles ( arrows ) . Scale bar : 50 μm .

O B-TISSUE O O B-DISEASE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O

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.


B , Immunoblotting of TLR7 in endosome isolated from HEK293T that were transfected with GFP - TLR7 or GFP - TLR7 mutant vectors .

O O B-EXP_ASSAY O B-GENEPROD O B-SUBCELLULAR O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O

B, Immunoblotting of TLR7 in the endosome isolated from HEK293T cells that were transfected with GFP-TLR7 or GFP-TLR7 mutant vectors.


G HEK293T transfected with NF - κB - luciferase reporter indicated TLR7 mutant vectors then treated with IMQ ( 10 µg / ml ) for 24 h were harvested for luciferase . Data information : Data are represented mean ± . P values are determined by two - unpaired Student ' s t - * * P < 0 . 01 , ns , not significant .

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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 of skin sections from human . Scale bar : 100 μm .

O O O O B-GENEPROD O O B-TISSUE O O B-ORGANISM O O O O O O O

I Representative p-TLR7 Y1024 staining of skin sections from human. Scale bar: 100 μm.


Tlr7wt / wt mice ( n = 6 ) Tlr7 - Y1025D mutant Tlr7ki / wt mice ( n = 6 ) Tlr7ki / ki mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . A Phenotypic presentation ( top ) H & E ( bottom ) of dorsal skin . Scale bar : 100 μm . Left : H & E data ; right : statistical data ( mean ± ) . Data information : Data are represented mean ± . P values are determined by two - Mann - Whitney U * P < 0 . 05 , * * P < 0 . 01 .

B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. 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 mice ( n = 6 ) Tlr7 - Y1025D mutant Tlr7ki / wt mice ( n = 6 ) Tlr7ki / ki mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . D Quantitative PCR of mRNA encoding IL - 23 / IL - 17A axis cytokines other psoriasis - related cytokines in dorsal skin . Results were normalized to Gapdh expression . Data information : Data are represented mean ± . P values are determined by two - Mann - Whitney U * P < 0 . 05 , * * P < 0 . 01 .

B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-DISEASE O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 mice ( n = 6 ) Tlr7 - Y1025D mutant Tlr7ki / wt mice ( n = 6 ) Tlr7ki / ki mice ( n = 5 ) were treated with indicated dose of IMQ for 4 days . E Representative p - p65 of dorsal skin . Scale bars : 100 μm .

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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.


A Microarray of microRNA expression changes in newborn mouse lungs exposed to 21 % O2 vs . 85 % O2 , at post - natal day ( P ) 5 P14 .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-TISSUE O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

A 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 lung over course of normal ( 21 % O2 ) aberrant ( 85 % O2 ) alveolarization . C Quantitative RT - PCR detection of microRNA - 34a / b / c - 3p family members in lung over course of normal ( 21 % O2 ) aberrant ( 85 % O2 ) alveolarization .

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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.


A Qualitative of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) miR - 34a - / - mice during normal aberrant alveolarization ( scale bar , 50 µm ) . B of total number of alveoli by design - stereology in wild - type ( WT ) miR ‑ 34a - / - mice ( 34a - / - ) during normal aberrant alveolarization . C of mean septal thickness by design - stereology in wild - type ( WT ) miR ‑ 34a - / - mice ( 34a - / - ) during normal aberrant alveolarization .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O B-GENEPROD O O O O O O O O O

A 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 of lung structure in Richardson - stained plastic - embedded lung sections from wild - type ( WT ) miR - 34bc - / - mice during normal aberrant alveolarization ( scale bar , 50 µm ) . E of total number of alveoli by design - stereology in wild - type ( WT ) miR ‑ 34bc - / - mice ( 34bc - / - ) during normal aberrant alveolarization . F of mean septal thickness by design - stereology in wild - type ( WT ) miR ‑ 34bc - / - mice ( 34bc - / - ) during normal aberrant alveolarization .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-ORGANISM O O O O O O O O O O O

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 in developing lungs of P14 miR - 34a : : lacZ + / + mice that were undergoing normal or aberrant alveolarization ( scale bar , 50 µm ; larger some additional are presented in Appendix Fig S2 ) .

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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).


B Immunoblot detection of PDGFRα in MLg after treatment with scrambled microRNA ( SCR ) or a miR - 34a ( MIM34a ) mimic ( n = 3 separate cultures for each group ) .

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B 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 in MLg in vitro , maintained under 21 % O2 or 85 % O2 ( n = 3 separate cultures for each group ) .

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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 in lungs of mouse pups ( n = 6 for each group ) at post - natal day ( P ) 5 , during normal ( 21 % O2 ) aberrant ( 85 % O2 ) alveolarization .

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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 in MLg in vitro , maintained under 21 % O2 or 85 % O2 , where had been transfected wither with a scrambled ( SCR ) antimiR , or an antimiR directed against miR - 34a ( A34a ) ( n = 3 separate cultures for each group ) .

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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 in PDGFRα + , sorted by FACS from lungs of mouse pups ( n = 4 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 .

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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 in PDGFRα + , sorted by FACS from lungs of either wild - type ( 34awt / wt ) mouse pups , or mouse pups in which miR - 34a expression in Pdgfra ‑ expressing ( 34ai ∆ PC / i ∆ PC ) at P5 ( n = 4 for each group ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O B-TISSUE O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O

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 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 of total number of alveoli by design - stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal aberrant alveolarization ( n = 5 for each group ) . K of mean septal thickness by design - stereology in 34awt / wt or 34ai ∆ PC / i ∆ PC mouse pups at P14 , during normal aberrant alveolarization ( n = 5 for each group ) .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O

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).


B Evaluation of specificity of TSB1 TSB2 in MLg using scrambled miR ( SCR ) miR - 34a ( MIM34a ) mimics , probing for PDGFRα c - Kit as TSB ‑ dependent TSB ‑ independent target readouts , respectively . loading equivalence was controlled by βactin . Note : PDGFRα , βactin , c - Kit were probed on same ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O

B 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 specificity of TSB1 TSB2 in MLg using scrambled miR ( SCR ) miR - 34a ( MIM34a ) mimics , probing for PDGFRα SIRT1 as TSB ‑ dependent TSB ‑ independent target readouts , respectively . loading equivalence was controlled by βactin . Note : PDGFRα , βactin , SIRT1 were probed on same ; hence a single βactin immunoblot is presented . Data are representative of three experiments .

O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O

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 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 TSB2 ) during normal ( 21 % O2 ) aberrant ( 85 % O2 ) alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments . E of total number of alveoli by design - stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) . F of mean septal thickness by design - stereology in wild - type mouse pups at P14 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O 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-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O 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 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 of PDGFRα + by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O 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 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 of PDGFRα + / αSMA + by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with scrambled target site blocker ( SCR ) or TSB1 , 2 cocktail during aberrant alveolarization ( n = 5 for each group ) .

O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O 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 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).


B Quantitative RT - PCR detection of miR - 34a - 5p 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 ) aberrant ( 85 % O2 ) alveolarization ( n = 4 for each group ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O 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-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O

B 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 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 aberrant alveolarization ( scale bar , 50 µm ) . Data are representative of three or more experiments .

O O O B-TISSUE B-EXP_ASSAY O O O O O O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

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 of total number of alveoli by design - stereology in wild - type mouse pups at post - natal day ( P ) 14 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal aberrant lung alveolarization ( n = 5 for each group ) .

O O O B-EXP_ASSAY O B-TISSUE O O O B-EXP_ASSAY O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-TISSUE O O O O O O O O O O

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 of mean septal thickness by design - 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 aberrant alveolarization ( n = 5 for each group ) .

O O O B-TISSUE B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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 of PDGFRα + by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal aberrant alveolarization ( n = 5 for each group ) .

O O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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 of PDGFRα + / αSMA + by flow cytometry , in lungs from wild - type mouse pups at P5 , treated with either scrambled antimiR ( S ) , or antimiR - 34a ( A34a ) , during normal aberrant alveolarization ( n = 5 for each group ) .

O O O B-GENEPROD O O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

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).