Extended Data Fig. 2: SOX2 maintains pluripotency of SOX2-TetON ES cells.
(a) Flow cytometry analysis shows that SOX2-TetON cultured in the presence of doxycycline (SOX2-ON) express SOX2 at comparable levels to pluripotent stem cells. (b) Brightfield migrograph showing that SOX2-ON maintain an undifferentiated morphology in ‘2i’ medium. (c) Flow cytometry analysis shows that SOX2 levels are progressively reduced following removal of Dox from SOX2-TetON (SOX2-OFF). (d) Brightfield migrograph showing that SOX2-OFF cultured in ‘2i’ medium lose their undifferentiated morphology. (e) Immunofluorescence image showing that SOX2-OFF cultured in ‘2i’ lose expression of pluripotency markers OCT4 and NANOG and (f) induce the primitive streak marker T/BRA in the absence of SOX2 expression. Images shown in figures B, D, E, F are representative of 3 independent experiments. Scale bars represent 75uM. (g) Measurement of relative mRNA expression by RT-qPCR shows that SOX2-OFF induce expression of the EpiLC marker Sox3 at similar levels to WT EpiLCs. Measurement of relative mRNA expression by RT-qPCR shows that ablation of Sox3 in SOX2-OFF (SOX2-OFF, SOX3-) results in loss of expression of the pluriptency marker Pou5f1 (h) and EpiLC marker Fgf5 (i). (j) FACS analysis shows that SOX2 levels in SOX2-OFF are intermediate to SOX2 low CEpiLCs and SOX2 -ve paraxial mesoderm progenitors. (k) Measurement of relative mRNA expression by RT-qPCR shows that Sox3 levels are reduced in CHIR-stimulated SOX2-OFF compared to WT CEpiLCs. Each data point in G, H, I, and K represents an individual biological replicate. Bars denote mean ± s.e.m. P values calculated for differences of mean expression by two-tailed Student’s t-test are shown. In G n=12 for ESC, n=4 for EpiLC, and n=4 for SOX2-OFF samples. In H n=6 for EpiLC, n=4 for SOX2-OFF, and n=6 for SOX2-OFF, SOX3- samples. In I n=10 for EpiLC, n=4 for SOX2-OFF, and n=6 for SOX2-OFF, SOX3- samples. In K n=4 for CEpiLC, and n=4 for SOX2-OFF samples. Source numerical data are available in source data.
