Fig. 3

NPRL2 depletion impedes proliferation of the oncogenic HRas-transformed cells. A NPRL2 depletion reduces the proliferation of Ras-AI-T2 cells. Ras-AI-T2 cells were transduced with control or NPRL2-targeting shRNA-containing virions (10 or 20 μL virion-containing medium/10⁴ cells) for 48 h. Then, the cells were replated, and the viable cell numbers were monitored for 96 h at 24-h intervals with trypan blue dye exclusion assay. Results are the mean ± SD from three experiments. ***p < 0.001 by two-way ANOVA. B, C NPRL2 depletion reduced colony formation ability of Ras-AI-T2 cells. Ras-AI-T2 cells were transduced with control or NPRL2-targeting shRNA-containing virions (10 or 20 μL virion-containing medium/10⁴ cells) for 48 h. Then, the cells were subjected to colony formation (B) or soft agar colony formation (C) analyses. Results are the mean ± SD from at least three experiments. *p < 0.05 and **p < 0.01 by Student’s t-test. D NPRL2 depletion reduces the ability of Ras-AI-T2 cells to form xenografted tumors. Ras-AI-T2 cells were transduced with control or NPRL2-targeting shRNA-containing virions (10 or 20 μL virion-containing medium/10⁴ cells) for 48 h. Then, the cells were collected and used for xenograft tumor formation assays (3–4 mice in each group). Tumor volumes (upper panels) are shown for individual mice (points) and the mean for each group (line). ***p < 0.001 by two-way ANOVA. Body weights (mean ± SD) are shown in lower panels and did not differ between groups. E Rapamycin reduces NPRL2 depletion-induced cell death and apoptosis. Left, Ras-AI-T2 cells were transduced with control or NPRL2-targeting shRNA-containing virions (20 μL virion-containing medium/10⁴ cells) for 48 h. Then, the cells were replated in the absence or presence of 20 nM rapamycin for 72 h, and the viable cell numbers were measured with trypan blue dye exclusion assay. Alternatively, the cells were subjected to flow cytometry analysis of annexin V-positive cells (right). Results are the mean ± SD from three experiments. **p < 0.01 by Student’s t-test