Fig. 2

Depletion of NPRL2 activates mTORC1 downstream signaling and inhibits autophagy in oncogenic HRas-transformed cells. A The NPRL2 mRNA expression level in Ras-AI-T2 cells transduced with virions containing empty vector (pLKO.1) or shRNA-targeting NPRL2 (shNPRL2). Forty-eight hours after transduction (20 μL virion-containing medium/10⁴ cells), the level of NPRL2 was analyzed by qPCR. Data are the mean ± SD from three independent experiments. ***p < 0.001 according to Student’s t-test. B NPRL2 depletion activates mTORC1 and inhibits autophagy. Left, Ras-AI-T2 cells were transduced with control or NPRL2 shRNA containing virion (20 μL virion-containing medium/10⁴ cells) for 48 h or 96 h. Then, immunoblotting analyses were performed to measure the levels of NPRL2, mTORC1 downstream substrates, and autophagy markers. Right, Ras-AI-T2 cells were transduced as described above and treated with 50 μM chloroquine (CQ) for 96 h. Immunoblotting analyses were performed to measure the level of autophagy markers. C NPRL2 depletion increased the percentage of cells with puncta that were LC3-positive and LAMP1-negative. Left, representative images of control and NPRL2-depleted Ras-AI-T2 cells stained for LC3 (green) and LAMP1 (red). Nuclei were counterstained with DAPI (blue). Scale bar, 10 μm. Arrowheads indicate the cells with puncta stained only for LC3 and not LAMP1. Right, the percentages of cells displaying puncta stained only for LC3 but not LAMP1. Results are the mean ± SD from at least 200 cells for each condition, as determined from three experiments. *p < 0.05 according to Student’s t-test