Fig. 6. RSL24D1 is required for mouse ESC proliferation and self-renewal.

a Analysis of proliferative capacities defined by the cell confluence (Incucyte® technology) of CGR8 ESC^FBS treated with si-CTL or si-Rsl24d1, without or with doxycycline-induced RSL24D1 rescue (n = 3). Data are presented as mean values +/− SEM. Multiple paired t test. b FACS analysis of EdU incorporation and DNA content (FXCycle Violet) in ESC^FBS treated with si-CTL or si-Rsl24d1, with or without RSL24D1 protein rescue. The percentage of cells in the different phases of cell cycle are indicated. Two tailed Student’s t test. c Analysis of the self-renewing capacities of CGR8 ESC^FBS estimated by the quantification of the number of individual colonies with alkaline phosphatase activity detected by colorimetric labeling (upper graph) and the relative alkaline phosphatase activity in each colony (lower graph) (n = 4). Representative images of the ESC colonies are shown for the si-CTL (gray frame), si-Rsl24d1 without (black frame) or with (red frame) RSL24D1 protein rescue conditions. Two tailed Student’s t test. Scale bars represent 400 μm. d Heatmap representation of the expression profiles of 60 marker genes in si-CTL, si-Rsl24d1, and si-Rsl24d1-rescue CGR8 cells cultured with LIF (Day 0) or in absence of LIF with retinoic acid for 4 days to induce differentiation (Day 4). e Dot plots representing pluripotency, ectoderm, endoderm, and mesoderm gene signatures, as previously defined⁴³. Each biological replicate (n = 3) is indicated by an individual dot and the average score for each condition by a bar. Multiple paired t test: n.s. p > 0.05, *p < 0.05, **p < 0.01, ****p < 0.0001. f Analysis of the proportion of 12-day old EBs, generated from CGR8 cells expressing control- (n = 7), Pou5f1- (n = 5) or Rsl24d1-shRNAs (n = 7), containing functional self-beating cardiomyocytes. Two tailed Student’s t test.