Fig. 2.

Rbm47 depleted mESCs are pluripotent and can self-renew. A-B Rbm47 knockdown efficiency by indicated shRNAs at RNA (Error bar represents the S.E.M.) and protein levels from three biological replicates. shlacZ was used as a non-targeting control shRNA. Relative RBM47/TUBB protein quantification is represented as mean ± S.E.M from three independent blots. C Phase-contrast images of shlacZ mESCs and shRbm47 mESCs, scale-100 μm, (Top). Cells were fixed and stained for alkaline phosphatase (ALP) activity, scale-100 μm, (Bottom). D ALP activity was quantified by calculating integrated densities (I.D) of images processed in ImageJ software. Mean integrated densities ± S.E.M from three staining experiments were plotted. E Relative mRNA expression of indicated markers in control and shRbm47 mESCs. Log₂ normalized values from three biological replicates were used for heatmap generation. F Western blot analysis of indicated pluripotency markers. Relative quantification is represented as mean ± S.E.M from three independent blots. TUBB was used as the loading control. G Widefield fluorescence images of mESCs stained for pluripotency markers (Scale-100 μm). H Cell cycle profiling of control and shRbm47 mESCs from three biological replicates. The graph represents mean percent population ± S.E.M. Statistical test used for A, B, D, F and H: unpaired student’s t-test; ns- non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001