Fig. 3: hESC^1q have an MDM4-driven competitive advantage that is retained during differentiation.

A Representative images of immunostaining for PAX6 (magenta left panel), HNF4A (magenta, middle panel) and GATA4 (magenta, right panel) after induction of cell competition during the differentiation to neuroectoderm, hepatoblast and cardiac progenitors. Throughout all panels, 1q cells are shown in yellow, POU5F1 cells in turquoise and DNA in white. B Quantification of 1q-cells at the start and end of differentiation. C Western blot and intensity quantification of protein bands of MDM4 and p53 in hESC^wt, hESC^1q and siMDM4-treated hESC^1q. D Time-course of mRNA expression of MDM4 in siMDM4-treated and untreated hESC^1q. E Images of the competition assays in the undifferentiated state. hESC^1q (green) are mixed in at a 1:9 ratio with hESC^wt and traced by their fluorophore expression. In the siMDM4 condition, the hESC^1q have been treated with siRNA against MDM4 prior to mixing. F Images of the competition assays of hESC^20q11.21 (red) in the undifferentiated state. The experimental setup is as in panel (E). G Quantification of hESC^1q at the start of the cell competition (day 0) and at day 3 of in vitro culture. H Quantification of the numbers of hESC^20q11.21 at the start of the cell competition (day 0) and at day 3 of in vitro culture. I Quantification of 1q-cells at the start of the cell competition during neuroectoderm, hepatoblast and cardiac progenitor differentiation (day 0) and at day 3 or 4, with and without siRNA against MDM4. J Representative images of immunostaining for PAX6 (magenta left panel), SOX17 (magenta, middle panel) and GATA4 (magenta, right panel) after induction of cell competition during the differentiation to neuroectoderm, hepatoblast and cardiac progenitors, with and without treatment of the hESC^1q with siRNA against MDM4. Throughout all panels, 1q cells are shown in yellow, POU5F1 cells in turquoise and DNA in white. K Quantification of 1q-cells in embryoid bodies after 4 days of spontaneous differentiation and directed neuroectoderm differentiation. Four EBs were pooled for dissociation and counting of fluorescently labeled 1q cells. L Example of embryoid bodies for cell line VUB19 on day 1 and 4 of spontaneous and directed neuroectoderm differentiation. Cells with 1q gain are labeled with green fluorescence. *p < 0.05, **p < 0.01, ****p < 0.0001, ns = non-significant.