Fig. 4: p63 is essential for the pro-invasive SMAD-AP-1 program.

a qRT-PCR analysis to investigate the role of p63 in TGFβ + EGF-induced gene expression. MCF10A MII cells were transfected with non-targeting control (siNTC) or specific p63 siRNA, serum-starved for 16 h, and stimulated for 1.5 or 16 h with TGFβ1 (5 ng/ml), as indicated. Statistics were calculated using one-way analysis of variance (ANOVA). The data were further analyzed using Tukey’s multiple comparisons test. Results from four independent experiments are shown as mean ± SD; ns, not significant, **P < 0.01, ***P < 0.001. b qRT-PCR analysis to investigate the effect of p63 depletion on TGFβ + EGF-induced target genes. HCC1954 cells were transfected with non-targeting control (siNTC) or specific p63 siRNA, serum-starved for 16 h, and stimulated for 1.5 (SMAD7) or 16 h with TGFβ1 (5 ng/ml), as indicated. Statistics were calculated using one-way analysis of variance (ANOVA). The data were further analyzed using Dunnett’s multiple comparisons test and compared with the results from cells transfected with non-targeting control (siNTC) and treated with TGFβ1 (5 ng/ml). Results from three independent experiments are shown as mean ± SD; **P < 0.01, and ***P < 0.001. c Effect of p63 knockdown on collagen-invasion of MCF10A MII spheroids in the presence or absence of 5 ng/ml TGFβ1 and 20 ng/ml EGF, as indicated. Cells were transfected with non-targeting control (siNTC) or specific p63 siRNA before spheroid formation. Left: relative invasion was quantified as the mean area that the spheroids occupied 24 h after being embedded in collagen. Statistics were calculated using one-way analysis of variance (ANOVA). The data were further analyzed using Tukey’s multiple comparisons test. Data represent mean ± SD (n ≥ 6 spheroids per condition) and are representative of three independent experiments; ns, not significant, *P < 0.05, and ***P < 0.001. Right: representative pictures of spheroids were taken 24 h after embedding. d The effect of p63 knockdown on SMAD2/3 recruitment by TGFβ and EGF. ChIP-qPCR showing SMAD2/3 binding to the indicated gene loci in MCF10A MII cells transfected with non-targeting control (siNTC) or specific p63 siRNA, serum-starved with or without EGF (20 ng/ml), and stimulated for 6 h with 5 ng/ml TGFβ1 (5 ng/ml) or untreated, as indicated. One of two independent experiments with similar results, is shown. e p63 interaction with SMAD2/3 and JUNB. MCF10A MII cells grown in the presence of EGF (20 ng/ml) were stimulated with TGFβ (5 ng/ml, 45 min) or not, and whole cell lysates (WCL) were immunoprecipitated (IP) with JUNB or SMAD2/3 specific antibodies, or IgG control, and analyzed by immunoblotting. One of three independent experiments with similar results, is shown.