Figure 4 | Cell Death & Disease

Figure 4

From: ARHI (DIRAS3)-mediated autophagy-associated cell death enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts

Figure 4

ARHI re-expression induced necrosis and reactive oxygen species (ROS) production as well as RIP1-mediated necroptosis in ovarian cancer cells. (a) ARHI-induced autophagy induces necrosis in ovarian cancer cells. SKOv3-ARHI cells were treated with DOX to induce ARHI expression at the intervals indicated. Live cell staining was performed with a combination of Hoechst 33342 BF (blue) and PI (red) dyes to determine the nuclear morphology and membrane integrity judged by immunofluorescence microscopy. Scale bars: 10 μm. The columns indicate the mean, and the bars indicate the S.E. (**P<0.01; DOX+ versus DOX−). (b) Re-expression of ARHI induced ROS that depended upon autophagy. SKOv3-ARHI, SKOv3-ARHI-shControl and shATG5 cells were treated with DOX to induce ARHI expression at the intervals indicated and cell lysates and supernatant were collected for ROS detection. The figure shows the combined values of two independent experiments. The columns indicate the mean, and the bars indicate the S.E. (**P<0.01; shATG5 versus shCtrl). (c) ARHI interacts with RIP1 and RIP3. To examine the interaction with RIP1 and RIP3, SKOv3-ARHI cells were treated with or without DOX. Endogenous RIP1/RIP3/FADD/caspase-8/ARHI/LC3 complexes were immunoprecipitated with anti-RIP1 antibody and analyzed for co-immunoprecipitation of RIP1/RIP3/FADD/caspase-8/ARHI/LC3 conjugates (IP). Host species-matched nonspecific IgG served as negative controls. Whole-cell lysates (WCLs) are included for comparison. (d) Necrostatin-1 (Nec-1) significantly rescued ARHI-induced loss of cell viability. SKOv3-ARHI and Hey-ARHI cells were treated with DOX and Nec-1 (40 μM) simultaneously at indicated times. Cell viability was measured with SRB assay. The figure shows the combined values of three independent experiments. The columns indicate the mean, and the bars indicate the S.E. The numbers indicate the ratio of DOX− versus DOX+ and ratio of DOX− Nec-1 versus DOX+ Nec-1. Differences of ratio between Nec-1 treatment and no treatment were considered statistically significant at *P<0.05 and **P<0.01

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