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. 2015 Jun 15;29(12):1239–1255. doi: 10.1101/gad.257071.114

Figure 3.

Figure 3.

Gata6 induction results in dynamic changes in cell morphology and gene expression even in the absence of Oct4 and Fgf4. (A) Representative phase-contrast images of Gata6-overexpressing mES cells at defined time points from 0 to 144 h of doxycycline treatment. Bars, 100 μm. (B) qRT –PCR analysis for selected pluripotency and endoderm transcripts in Gata6-overexpressing mES cells between 0 and 144 h of doxycycline induction. Relative expression is reflected as fold difference over uninduced mES cells normalized to Gapdh. Data are mean ± SEM of three biological replicates. (C) Western blot for selected proteins in Gata6-overexpressing cells from 0 to 144 h of doxycycline induction. A representative Actin loading control is included as a reference. Endogenous (endo) and exogenous (exo) Gata6 bands are indicated. (D) Western blot for selected proteins in Sox17-overexpressing cells from 0 to 144 h of doxycycline induction. A representative Actin loading control is included as a reference. (E) qRT –PCR analysis for selected pluripotency and endoderm transcripts following 72 h of shRNA knockdown of Pou5f1 during Gata6 induction compared with scrambled control shRNA. Data are mean ± SEM of five distinct shRNA constructs and two biological replicates. (***) P < 0.001. (F) Western blot for selected proteins in Pou5f1 knockdown cells at the time points indicated. A representative Actin loading control is included as a reference. (G) Phase-contrast images of Oct4 conditional knockout cells following 6 d in the absence or presence of exogenous Gata6 induction. Bars, 20 μm. (H) Western blot for Oct4 protein in Oct4 conditional knockout cells at the time points indicated in the presence or absence of exogenous HA-tagged GATA6. A representative Actin loading control is included as a reference. (I) Phase-contrast image of stable Fgf4−/− iXEN cells. Bars, 20 μm. (J) Western blot for selected proteins in Fgf4-null mES cells following 6 d of Gata6 induction in the absence or presence of Fgf4 (F) and heparin (H). A representative Actin loading control is included as a reference.