Fig 6. Wnt-dependent cell cycle regulation in mESCs is due to Tcf1 activity.

(A) Cell number quantification of control and Knock-Out Tcf1 ESC clones treated with indicated BIO concentrations for 24 and 48h (n = 3). The asterisks indicate statistical significance by two-way ANOVA analysis. Statistical significance is shown: (i) between same cell types at different time-points of BIO treatment (as *); and (ii) between different cell types at the same BIO treatment concentration (as #). (n.s. not significant; * or ^# p<0.05; ** or ^## p<0.01; *** or ^### p<0.001). (B) qRT-PCR experiment for Tcf1 cell cycle target genes (p15^Ink4b, p16^Ink4a, p18^Ink4c, p19^Arf) in control and KO Tcf1 clones (clones: C1, C7 and C8) treated with BIO for 48h. (n = 6, two-tailed Student’s t-test analysis, BIO treated cells compared to respective DMSO treated cells.). (C) Representative Western blot of β-catenin, p16^Ink4a, p19^Arf and β-actin in 72h BIO-treated WT and Tcf1 KO cells. (D) Cell growth curve of dCas9-Vp64-eGFP transduced mESCs, additionally transduced with the empty vector without any sgRNA and sgRNA Tcf1A1 constructs (sgRNATcf7; see S7 Fig) in Serum+LIF conditions (n = 2). (E) Cell number quantification of control and Tcf1 overexpressing mESC clones (sgRNATcf7) treated for 2 and 5 days with DMSO (0,15%) and 3 μM of BIO (n = 2). (F) Representative Western blot of β-catenin, Tcf1, p16^Ink4a, p19^Arf and β-actin in 48h BIO-treated WT and Tcf1-OE cells. All pooled data are represented as means ± SD.