Fig. 5

p38α modulate β-catenin target gene expression. A Chromatin immunoprecipitation assays of Wnt target genes in HT-29 cells under serum starvation (24 h) and upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with the p38α inhibitor ralimetinib (10 μM) for 24 h. Quantification was done using the % input method. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. untreated cells, and ^#P < 0.05 vs. no ralimetinib. B RTqPCR analysis of Wnt target genes in HT-29 cells upon activation of the Wnt pathway mediated by the addition of Wnt3a (50 ng/mL) and the GSK3β inhibitor TWS-119 (10 μM) for 4 h after p38α genetic ablation for 24 h or as a pre-treatment before p38α inhibition with ralimetinib (10 μM) for 24 h. Data are presented as mRNA fold change vs. control. The dotted line corresponds to the expression levels detected in control conditions (siRNA CTRL/DMSO). Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. siRNA CTRL/DMSO. C TOPFlash/FOPFlash assay for Wnt transcriptional activity. HT-29 cells were first transfected to overexpress p38α and β-catenin; after 24 h, cells were transfected with TOP/FOP plasmids, serum-starved for 24 h and then stimulated with Wnt3a (50 ng/mL) and TWS-119 (10 μM) for 4 h. Subsequently, cells were treated or not with ralimetinib (10 μM) for 24 h. Statistical analysis was performed using Student’s t-test: *P < 0.05 vs. empty vector, ^#P < 0.05 vs. DMSO, ^▲P < 0.05 vs. no serum. Results are representative of at least three independent experiments