A is required for proliferation and differentiation of ESC+2i A.Figure 1A. BAZ2A expression is higher in ESC+2i than in differentiated cells (neural progenitors, NPC). Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Right panel. Western blot showing BAZ2A protein levels in ESC+2i and NPC. Tubulin is shown as a protein loading control. B. BAZ2A associates with chromatin of ESC and NPC. Chromatin-bound (Chrom.) and soluble (Sol.) fractions of equivalent cell number of ESC+2i and NPCs were analyzed by western blot for BAZ2A levels. Total, total protein. Tubulin and histones are shown as loading and fractionation control. C. siRNA-knockdown efficiency of BAZ2A shown by qRT-PCR and western blot. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to each ESC line. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (**** < 0.0001). Right panel. Western blot showing BAZ2A protein levels. Tubulin is shown as a protein loading control. D. BAZ2A knockdown affects proliferation of ESC+2i but not of ESC+serum. Data represent relative cell numbers after 3 days of siRNA treatment and were normalized to ESC transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (** < 0.01; ns, non-significant). E. BAZ2A is expressed at similar levels in both ESC+2i and ESC+serum. Left panel. BAZ2A mRNA levels were measured by qRT-PCR and normalized to Rps12 mRNA and to ESC+2i. Average values of three independent experiments. Error bars represent s.d. Right panel. Western blot showing BAZ2A protein levels in ESC+2i and ESC+serum. Tubulin and histone H3 are shown as a protein loading controls. F. BAZ2A is required for the differentiation of ESC+2i but not of ESC+serum. Representative images of alkaline phosphatase staining of ESC and cells after 3 days of differentiation upon culture in completed medium containing 10% serum and in the absence of LIF. G. Quantification of differentiated cells. Values represent relative number of differentiated cells from three independent experiments relative to control cells. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (*** < 0.001; ns, non-significant). H. Western blot showing BAZ2A protein levels in two BAZ2A-KO ESC lines obtained via CRISPr/Cas9 directly in ESC+serum. PARP1 is shown as a protein loading control. I. Cell proliferation is not affected in BAZ2A-KO ESC+serum. Data represent relative cell numbers 2 days culture starting with the same number of cells. Average values of four independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns, non-significant). J. Representative images of wt and Baz2a-KO ESC+serum before and after transition in 2i conditions.