Figure 3

Characterization of iOCT4 ES cells cultured in LIF-independent ES cell media (A) Dapi cell cycle staining of wild-type ES cells (+LIF/+GSK3i) and iOCT4 LIF-independent iOCT4 ES cells (−LIF/+GSK3i/−dox) cultured in FBS-containing ES cell media. (B) Western blot of OCT4 and SOX2 (left and middle), and STAT3 and STAT3-pY705 (right), in control (ZHTc6) ES cells and LIF-independent iOCT4 ES cells cultured in FBS-containing media and GSK3i. HSC70 or ACTIN were used as loading controls. (C) Western blot of OCT4 and SOX2 (top), and STAT3 and STAT3-pY705 (bottom), in wild-type (R1) and LIF-independent iOCT4 ES cells cultured in serum-free ES cell media without GSK3i or doxycycline. (D) Western blot of OCT4 (top), and STAT3 and STAT3-pY705 (bottom), in LIF-independent iOCT4 ES cells cultured without GSK3i or LIF and with doxycycline to repress OCT4 transgene expression over a time-course of 6 days. Note that the level of OCT4 protein is significantly decreased after three days of culture in the presence of doxycycline. (E) Western blot of OCT4 in wild-type (R1) ES cells cultured in FBS-containing ES cell media with or without LIF, and with or without GSK3i for 11 days. Note that the level of OCT4 protein is significantly reduced in wild-type ES cells cultured in the absence of LIF. (F) Immunofluorescence analysis of SOX2, DPPA2, OCT4, and SSEA1 in control ES cells and LIF-independent iOCT4 ES cells cultured in FBS-containing ES cell media without LIF, GSK3i, or doxycycline. Nuclei were stained with Dapi. (G) Giesma staining of metaphase spreads and chromosome counting revealed that iOCT4 LIF-independent ES cells cultured in FBS-containing media and GSK3i have a normal karyotype.