Fig. 4

Circadian control of EMT by HNF4α is isoform specific. a RT-PCR reveals mRNA abundance of EMT genes CDH1, CTNNB1, SNAI1, and SNAI2 after serum shock with prior application of scrambled oligonucleotides or siRNA specific to P1-HNF4α in HepG2 cells. b Western blot showing expression of CDH1, phosphorylated, and total β-catenin (CTNNB1, β-Cat), after P1-HNF4α knockdown followed by serum shock. c RT-PCR reveals expression of EMT genes following serum shock and prior application of scrambled or siRNA specific to P2-HNF4a. d Western blot showing expression of CDH1, phosphorylated and total β-Cat following knockdown of P2-HNF4α and serum shock. e RT-PCR reveals circadian expression of EMT genes CDH1, CTNNB1, and SNAI1 after inhibition of P2-HNF4α using specific siRNA oligonucleotides in SNU449 cells. f Western blot showing the expression of CDH1 and phosphorylated and total β-Cat after P2-HNF4α knockdown following serum shock in SNU449 cells. Two-way ANOVA, Sidak’s multiple comparisons test, ^*P < 0.03, ^**P < 0.005, ^***P < 0.0005, ^****P < 0.0001, (N = 4). g Invasion assay reveals invaded unsynchronized or circadian synchronized HepG2 cells expressing scrambled or siRNA for P1/P2-HNF4a, 48 h after plating. Quantification, right panel. h Invaded unsynchronized or synchronized Hepa-1c1c7 cells following serum synchronization and prior overexpression of P1-HNF4α. Quantification, right panel. i Invaded synchronized HepG2 cells following application of scrambled (“Sc”), P1-HNF4a, or P2-HNF4a siRNA oligonucleotides. Quantification, right panel. j Invaded synchronized Hepa-1c1c7 cells following overexpression of P1-HNF4α or P2-HNF4α. Quantification, right panel. Compared to SC or EV at the same time: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, one-way ANOVA test, Dunnett’s multiple comparisons test. (N = 5). k Fold change in proliferating HepG2 cells following P1-HNF4α vs. P2-HNF4α knockdown at 24- and 48 h using MTT assay. l MTT assay reveals proliferating Hepa-1c1c7 cells after transfection with empty vector (EV), P1-Hnf4a (Hnf4a2) or P2-Hnf4a (Hnf4a8) at 24- and 48 h using MTT assay. Comparing SC/EV to P1/P2-siHNF4α or between P1-siHNF4α and P2-siHNF4α: Two-way ANOVA, Sidak’s multiple comparisons test, ^*P < 0.03, ^**P < 0.005, ^***P < 0.0005, ^****P < 0.0001, (N = 6). Scale bar 100 µm. (See Supplementary Table 1 for JTK_Cycle Rhythmicity Statistics.) Error bars = SEM