Undifferentiated states of SNL- and MEFP1-201B7 cells.
(A and B) Quantification of mouse LIF and ActA protein concentrations in the culture media of SNLs and MEFs. The concentrations of mouse LIF (A) and ActA (B) protein in the collected culture media of SNLs and MEFs from days 1 to 6 (n = 4 each) were measured. Data represent the mean ± SEM. n.d.: not detected. *p < 0.05, **p < 0.01, or ***p < 0.001 versus SNLs. (C) The ALP activities of SNL- or MEFP1-201B7 colonies were analyzed by ALP staining. Black scale bar, 100 μm. (D and E) The expressions of the undifferentiated stem cell marker proteins OCT4, SOX2, SSEA4, TRA-1-81, and TRA-1-60 (red or green) in SNL- (D) or MEFP1-201B7 (E) colonies were analyzed by immunofluorescent staining. Cell nuclei were stained using DAPI (blue). White scale bar, 100 μm. (F) RT-qPCR analysis of undifferentiated stem cell marker gene expression in SNL- and MEFP1-201B7 cells. SNL- and MEFP1-201B7 cells were seeded in Matrigel-coated plates after the removal of feeder cells, and after a 24-hr incubation, total RNA was extracted from these cells and used to synthesize cDNA. The synthesized cDNA was used as a template for PCR. Human GAPDH-specific primer and RT-negative sample were used as positive and negative controls, respectively. PCR products were analyzed by agarose electrophoresis and stained with ethidium bromide. Data are representative of three experiments.