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. 2021 Feb 8;6(3):e142299. doi: 10.1172/jci.insight.142299

Figure 3. Overexpression of constitutively activated HRAS/KRAS diminishes NGBR deficiency caused G1 phase arrest.

Figure 3

(A and B) siControl and siNGBR cells were transiently transfected with empty vector or HRAS-G12V/KRAS-G12V expression vector for 24 hours. Protein levels of NGBR, p21, p53, cyclin D1, RB1, and phosphorylated RB1 were determined by Western blot. Quantitative analysis of proteins was carried out using ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. siNGBR cells (n = 3). (C) siControl and siNGBR cells expressing the FUCCI fluorescent CDT1-RFP (red) and geminin-GFP (green) were transiently transfected with HRAS-G12V or KRAS-G12V expression vector for 24 hours. The images were taken with a confocal microscope. Quantitative analysis of G1 phase cells was determined by ImageJ software. *P < 0.05 vs. control (siControl) cells. #P < 0.05 vs. siNGBR cells (n = 4), 3 repeats. Statistical analyses: 1-way ANOVA with Bonferroni’s post hoc test; data are expressed as mean ± SEM. NGBR, NOGOB receptor; siControl, control siRNA; siNGBR, NGBR siRNA; RB1, retinoblastoma; FUCCI, fluorescence ubiquitination cell cycle indicator.