Journal: Cell Death & Disease

Article Title: p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming

doi: 10.1038/cddis.2017.432

Figure Lengend Snippet: 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

Article Snippet: For experiments with P19 and H1299 cells involving BMP4 treatment, cells were serum-deprived (0.2% FBS) for 24 h, before the treatment with 0.5 to 50 ng/ml human BMP4 (Peprotech, Rocky Hill, NJ, USA) as indicated.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Activation Assay, Clone Assay, Plasmid Preparation, Cell Culture, Control

Journal: Cell Death & Disease

Article Title: p73 is required for appropriate BMP-induced mesenchymal-to-epithelial transition during somatic cell reprogramming

doi: 10.1038/cddis.2017.432

Figure Lengend Snippet: Smad6 is a direct DNp73 transcriptional target. ( a–c ) P19 cells were transfected with either ( a ) TA-, DNp73 or vector control, or ( b , c ) Smad6 -promoter reporter system. After 18 h cells were serum-deprived for 24 h and then treated with BMP4. At the indicated time points samples were collected and analyzed. ( a ) Quantification of Smad6 expression level was analyzed by qRT-PCR. ( b and c ) Transcriptional analysis was performed with the reporter vector pGL2-m Smad6 -promoter (-3123)- luc, a BMP-responsive reporter construct that includes the p53-RE, together with the indicated expression vectors in the presence or absence of BMP4. ( c ) TAp73 (0.2 ug) was co-transfected with increasing amounts of DNp73 (0.2–0.6 μ g) before BMP4 treatment. Luciferase activity was normalized by the Renilla activity of the same lysate. Bars represent mean values±S.E.M of at least four experiments; ns: not significant. ( d ) ChIP analysis of H1299 cells cultured in 10% serum conditions were performed using isotypic-antibody (rabbit IgG), anti-HA or anti-DNp73 specific antibodies. Real-time PCR using specific primers to amplify p53-RE of the human or mouse Smad6 promoter, or p53-RE in the p21 Cip1 promoter as control, were performed and the data were normalized to input chromatin samples of each case and to IgG values=1. Additional control was performed immunoprecipitating with either rabbit IgG or anti-TAp73 and anti-DNp73 specific antibodies, and PCR amplifying the ChIP product with primers specific to a region at the vicinity of the identified p53-RE (1700 bp downstream of the p53-RE), but without homology to this site (H1299 control). Experiments were repeated four times per duplicate. Bars represent mean values±S.E.M; equal variance. Student's t -test was performed to evaluate statistical differences; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: For experiments with P19 and H1299 cells involving BMP4 treatment, cells were serum-deprived (0.2% FBS) for 24 h, before the treatment with 0.5 to 50 ng/ml human BMP4 (Peprotech, Rocky Hill, NJ, USA) as indicated.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Construct, Luciferase, Activity Assay, Cell Culture, Real-time Polymerase Chain Reaction