Fig. 2.

myc depletion disrupts mESC pluripotency and self-renewal. (A) Immunofluorescent staining for SSEA-1 (red) and DAPI (blue) in the cDKO-2 mESC line. Arrow marks a representative GFP+colony, negative for SSEA-1. (B) Flow cytometric analysis of SSEA-1 expression in DKO-2 mESC line. mESC were analyzed by FACS for SSEA-1 levels with levels gated and defined as follows: negative (equal to or below levels of expression of isotype specific negative control), low (bottom third), medium (middle third), and high (top third) of SSEA-1 levels. Error bars are standard deviations. N=3. Decreases in high and medium SSEA-1 staining and increases in low and negative SSEA-1 staining had p values of 0.002, 0.005, 0.002, and 0.01, respectively, in cDKO2 and p values of 0.01, 0.002, 0.004, and 0.006 in cDKO3, respectively. (C) Fluorescent and phase contrast images of WT and DKO mESC stained for AP. Arrows denote AP staining in representative GFP-Cre positive colonies. (D) Percentage of self-renewing colonies of WT and DKO mESC lines calculated after alkaline phosphatase assays. Percentage of AP positive colonies in GFP only transduced mESC lines was defined as 100%. The error bars are SD and the data is the mean from three biological replicates (n=3). (E) Percentage of GATA6 positive cells out of GFP positive mESC (GFP alone or GFP-CRE) quantified by immunofluorescent staining for GATA6. The error bars are SD and the data is the mean from three biological replicates (n=3). P values were calculated by two-tail t-test assuming equal variances throughout this figure.