Fig. 3: SMAD2/3 initiate a collective migration transcriptional program in response of TGF-β in a SMAD4-null PDAC context.
From: SMAD2/3 mediate oncogenic effects of TGF-β in the absence of SMAD4
a Immunoblot analysis of βIG-H3, LPXN and GAPDH on control (Ctrl), S2-KO, S3-KO, and S2/3-KO BxPC-3 cells treated or not with TGF-β for 24 h. One representative image out of 3 independent experiments is shown. b Scatterplot showing the top 25 enriched biological pathways using Gene Ontology (GO) database based on a list of 144 differentially expressed genes between control and S2/3-KO that are specific to the TGF-β treatment response at 24 h. Dots are colored according to the adjusted statistical probabilities (FDR) and sized by the count number of genes matching the biological process. Gene ratio corresponds to the relative number of input genes and known associated genes of the tested biological pathway. c Bright field microscopy images of control and S2/3-KO BxPC-3 cells after TGF-β treatment. Cells were treated for 5 days, to allow potential EMT transition. One representative image out of 3 independent experiments is shown. Black arrows indicate the presence of large lamellipodia detectable only in control cells in the presence of TGF-β. Scale bar = 30 µm. d Immunoblot of RAC-1-GTP, RAC-1, and GAPDH on control and S2/3-KO BxPC-3 cells treated or not with TGF-β for 15 min. Graphs represent RAC1-GTP mean values +/– SD (n = 3). e Images of Vinculin and phospho-Focal Adhesion Kinase (pFAKY397) immunofluorescence performed on control and S2/3-KO BxPC-3 cells treated or not with TGF-β for 8 h, to avoid complete wound closure. Insets represent enlarged views of regions to visualize focal adhesion (FA) in leader cells. Nuclei were counterstained in blue with DAPI. Scale bar (left-hand images) = 2.5 µm. Scale bar (enlarged merge images) = 5 µm. Mean number of mature focal adhesions per cell with enlarged lamellipodia are represented as a graph. Variance is indicated as SD. One-tailed Wilcoxon/Mann–Whitney test; ***p-value < 0.001; ns, not significant. f Immunoblot of Paxillin (PXN), phospho-Focal Adhesion Kinase (pFAKY397), Focal Adhesion Kinase (FAK), and GAPDH on control and S2/3-KO BxPC-3 cells treated or not with TGF-β for 24 h. One representative image out of 3 independent experiments is shown. Ratios of pFAKY397/FAK were quantified from 3 independent experiments and represented as graphs of mean values +/– SD.
