Figure 6.

p73 is a positive modulator of the BMP circuit required for the OSKM-induced BMP signaling during the initiation phase of reprogramming. (a and b) Analysis of the expression kinetics profile of markers during reprogramming process of WT and p73KO-MEFs. RNA samples were collected at the indicated days and expression analysis was performed by qRT-PCR: (a) Id1 (b) Smad6 (left panel). Expression of the indicated genes was normalized to 18S and set to 1 for non-transfected MEFs in each graph (t=0). (b) Quantification of Smad6 expression by qRT-PCR in MEFs (right panel). Expression was normalized to 18S. Analysis was performed with data from two independent experiments, with at least three biological replicates from the indicated genotypes, with two replicates per sample. Mean±S.E.M. are represented, equal variance. Student's t-test was performed to evaluate statistical differences. *P<0.05, **P<0.01, ***P<0.001. (c) Western blot analysis of BMP signaling cascade activation in WT and p73KO-iPSC clones. (d) Quantification of Id1, Smad6, TAp73 and DNp73 expression by qRT-PCR after BMP4 treatment (0–50 ng/ml BMP4) in serum-deprived P19 cells. (e–g) P19 cells were transfected with DNp73 expression plasmid and after 18 h, cells were serum-deprived for 24 h and then, treated with BMP4. At the indicated time points BMP cascade activation was analyzed by quantification of Id1 by qRT-PCR (e, left panel) or phospho-Smad1/5/8 expression by western blot assay (e, right panel). (f and g) The Tet-OFF inducible cell line H1299-DNp73 was cultured on serum conditions (e) or serum-deprived (g) before inducing DNp73 expression in the presence or absence of BMP4 (g). BMP cascade activation was analyzed by phospho-Smad1/5/8 expression by western blot assay. Equal amounts of total protein were loaded and β-actin serves as loading control. Note that HA (Y11) antibody detects the exogenous DNp73 protein expression. At least two independent experiments were performed with similar results