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. 2016 Sep 12;44(22):10603–10618. doi: 10.1093/nar/gkw788

Figure 1.

Figure 1.

PRMT7 and its catalytic activity are indispensable for the stemness of mESCs, and Oct4, Nanog, Klf4 and c-Myc protein levels are more reduced than their mRNA levels in PRMT7-depleted mESCs. (A) Microscopic and AP-staining images of shLuc-treated and PRMT7-depleted V6.5 mESCs. Five shPRMT7s (shPRMT7-4, -5, -6, -7 and -8) were used to deplete PRMT7. BF, bright field; AP, alkaline phosphatase; red scale bar, 100 μm. (B) The percentages of shLuc- or shPRMT7-treated V6.5 mESCs in G1, S and G2/M phase of the cell cycle. shLuc-treated cells were used as a control. (C) Rescue experiments of PRMT7-depleted mESCs with PRMT7 or its catalytic mutant (m.PRMT7). shPRMT7-7 or shPRMT7-8 plasmids were co-electroporated with expression plasmids encoding both GFP and either PRMT7 or its catalytic mutant. The GFP-expressing vector pCDH1-EF1-IRES-GFP (vector/GFP) was used to clone PRMT7 cDNA. Microscopic, green-fluorescent and AP staining images of indicated group of cells are shown (left panel). Western blotting was used to analyze the expression levels of PRMT7 or its mutant in eight different experimental conditions (right panel). (D) Analysis of Oct4, Nanog, Sox2, c-Myc and Klf4 mRNA levels in shLuc-treated and PRMT7-depleted V6.5 mESCs using quantitative RT-PCR. (E) The effect of ectopic expression of PRMT7 or its catalytic mutant (m.PRMT7) on Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels in PRMT7-depleted mESCs. Relative Oct4, Nanog, Sox2, c-Myc, Klf4 and Prmt7 mRNA levels were examined in eight different experimental conditions (see panel C for lane description). Red scale bar, 100 μm. (F and G) Western blot analysis of Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 levels in shLuc-treated and PRMT7-depleted V6.5 mESCs. Oct4, Nanog, Sox2, c-Myc, Klf4 and PRMT7 protein levels in shLuc-treated and PRMT7-depleted V6.5 mESCs were quantified (G). Data are presented as the mean ± SD of three independent experiments. P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) indicate statistically significant changes.