Novel Function of Lysine Methyltransferase G9a in the Regulation of Sox2 Protein Stability (original) (raw)
Fig 2
The expression level of G9a affects the amount of Sox2 protein in MCF7 cells.
(A) MCF7 cells were transfected with siNS or siG9a for 24 h, and G9a transcript levels were analyzed by real-time PCR by priming two positions of G9a ORF (G9aI and G9aII). mRNA levels were normalized to those of GAPDH. G9aI and G9a2 primer sets were designed to amplify 1299~1531 bp and 2326~2637 bp of G9a ORF, respectively. (B) The effect of G9a on the proliferation of MCF7 cells. MCF7 cells (1 × 104) were transfected with siNS or siG9a and then counted at the indicated times post-transfection. (C) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the transcription of Oct4 and Sox2 was quantitatively analyzed by real-time RT-PCR. (D) MCF-7 cells were transfected with siNS or siG9a for 24 h, and the effect of G9a knockdown on the amount of Oct4 and Sox2 protein was analyzed by immunoblot analysis. (E) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the transcription of Sox2 and Oct4 was quantitatively analyzed by real-time RT-PCR. (F) MCF-7 cells were transfected with control or G9a expression plasmids for 24 h, and the effect of G9a overexpression on the amount of Sox2 and Oct4 was quantitatively analyzed by immunoblot analysis. (G) Co-immunoprecipitation of endogenous Sox2 and G9a in MCF7 cells. Total protein extract of MCF7 cells was immunoprecipitated with Sox2 and then immunoblotted with G9a antibody. The reciprocal experiment was also performed in which total protein of MCF7 cells was immunoprecipitated with G9a and then immunoblotted with Sox2 antibody. The specific bands corresponding to the G9a and Sox2 was arrowed. Abbreviations: siNS, non-specific siRNA; siG9a, siRNA targeting G9a; C, control plasmid; OE, G9a-expressing plasmid; IP, immunoprecipitation; IB: immunoblotting. For quantitative analysis of the immunoblotting results, the mean density of the Sox2 or G9a bands was measured with Multi Gauge V3.0 software. The band density was then divided by the density of β-actin to obtain the normalized band density. *P < 0.05 and **P < 0.01 compared with the control group. Data are expressed as the mean ± SDS and are representative of three independent experiments.