Skip to main content
. 2020 Feb 15;17(1):112–131. doi: 10.20892/j.issn.2095-3941.2019.0164

Figure 6.

Figure 6

The results of analyzing microarray data regarding Myc function and downstream Myc-mediated cell cycle-associated proteins were further validated. (A) The knowledge-based interactome surrounding the regulation of Myc was examined using ingenuity pathway analysis (IPA) and overlaid with microarray data with a 1.5-fold change cut-off. (B) The expression levels of downstream cell cycle-associated proteins were further validated. First, Western blot confirmed that Myc and p53 were downregulated in SNRPG-silenced U87 cells compared with that in shCtrl-transduced cells. In addition, the knockdown of SNRPG in U87MG cells also decreased the expression of other cell cycle-associated proteins, such as CDK2 and CCNB1. (C) SNRPG controls the subcellular localization of Myc. The levels of Myc, histone H3, and tubulin in the cytoplasmic (Cy) and nuclear (Nu) fractions of cells expressing a scramble or SNRPG-targeted shRNA, as assessed by Western blot, are shown, n = 3. (D) Western blot analysis of Myc, tubulin, and histone in the nuclear and cytoplasmic fractions of control and shSNRPG-expressing U87MG cells incubated with 0 or 5 ng of leptomycin B (LMB, 8 h). (E) SNRPG suppression increases cell cycle arrest via a Myc-dependent pathway. The cell cycle distribution was confirmed in SNRPG knockdown cells and in lentivirus-infected U87MG cells overexpressing Myc. *P < 0.05, n = 3. (F–I) The levels of SNRPG correlated inversely with p53 expression in vitro and in vivo, which affected the sensitivity of GBM cells to TMZ. (F) Western blot analysis showed that p53 and its target genes, especially p21, were upregulated in SNRPG-silenced U87MG cells compared with those in shCtrl-transduced cells (mean ± SD, n = 3, **P < 0.001 vs. Ctrl or shCtrl). (G, left panel) Representative Western blot analyses of p53 nuclear/cytoplasmic expression in control and shSNRPG-transfected U87MG cells in vitro. Bar = 50 μm, n = 3. (G, right panel) Average subcellular distribution of p53 (nuclear and cytosolic protein expression) and total p53 expression in U87MG cells from U87 tumor cells grown as xenografts in the shCtrl and shSNRPG groups. **P < 0.01, n = 3. (H–I) SNRPG suppression increased the p53 levels via a Myc-dependent pathway. The protein levels (H) and mRNA levels (I) of p53 were confirmed in SNRPG knockdown cells and in lentivirus-infected U87MG cells overexpressing Myc. *P < 0.05 and **P < 0.01 vs. the control group; #P < 0.05 vs. the shSNRPG group, n = 3.