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. 2019 Sep 26;17(9):e3000453. doi: 10.1371/journal.pbio.3000453

Fig 2. Single-cell distribution patterns of absolute G1 length determine differentiation propensity of hESC populations.

Fig 2

(A) Immunoflow cytometry of PAX6 and GATA6 in H9 cells differentiated for 8 d by FGF2 deprivation (n = 4). (B) Propidium iodide staining analysis of H9 cells either in E8 or in mTeSR1 media and human dermal fibroblasts (n = 4 for E8 and mTeSR1, n = 3 for fibroblasts). (C) Histograms for G1 length of H9 cells grown either in E8 or in mTeSR1 (n = 112 for E8 and n = 114 for mTeSR1 pooled from three independent experiments); U test: p-value < 2.2 × 10−16, KS test: p-value < 2.2 × 10−16. (D-F) qPCR analysis of lineage markers in differentiated day 8 H9 cells overexpressing p21 (D), CDK4R24C (E), or CDK6R31C (F) (n = 4). Ctrl represents cells transduced with empty lentiviral vectors and treated with the same dose of doxycycline (1 μg/ml). Transgene expression was turned off at the onset of differentiation by doxycycline withdrawal. Error bars represent SD. *p < 0.01 (Student t test). Underlying data can be found in S1 Data. CDK, Cyclin-dependent kinase; Ctrl, control; Diff. differentiated; E8, Essential 8; FSC, forward scatter; FGF, Fibroblast growth factor; FL, fluorescence intensity; GATA6, GATA binding protein 6; HAND1, Heart and neural crest derivatives expressed 1; hESC, human embryonic stem cell; KS, Kolmogorov-Smirnov; OTX1, Orthodenticle homeobox 1; PAX6, Paired box 6; qPCR, quantitative PCR; Undiff., undifferentiated; ZBTB16, Zinc finger and BTB domain containing 16.