Figure 4

p73 is dispensable for iPSCs self-renewal and in vitro pluripotency, but p73-deficient cells have decreased pluripotency marker expression and might not have attained full stemness. Three clones for each genotype were analyzed. All the p73-defective clones displayed the altered phenotype (a–c) iPSCs of the indicated genotype were cultured under proliferating and non-differentiating conditions and analyzed. (a) Representative phase contrast images (Objective 5 × ; Scale: 250 μm) of iPSCs cultures corresponding to the 20th passage. (b) Confocal microscopy analysis of pluripotency markers Nanog (red) and SSEA-1 (green). DAPI was used to visualize nuclei. Objective 20 ×. Scale: 80 μm. (c) Quantification of Lin28 expression by qRT-PCR. Mean values±S.E.M. from duplicates of at least three clones per genotype from two independent experiments are shown, equal variance. Expression was analyzed by qRT-PCR, normalized to 18S. Student's t-test was performed to evaluate statistical differences *P<0.05, **P<0.01, ***P<0.001. (d) iPSCs lines of the indicated genotype were differentiated by embryoid body (EB) formation under differentiating conditions and lineage markers of three germ layers were analyzed: Tuj-1 (ectoderm, green), CD31 (mesoderm, red) and AFP (endoderm, red). At least three clones per genotype were analyzed. DAPI was used to visualize nuclei. Objective 20 ×. Scale: 80 μm