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. 2020 Jul 31;6(31):eaay9131. doi: 10.1126/sciadv.aay9131

Fig. 4. Cancer cells with high mTORC1 activity have increased dependency on RUVBL1/2 for survival.

Fig. 4

(A) The effect of RUVBL1/2 depletion on cell viability. Cell death was measured 4 days (T24) or 6 days (CCD-18co, HDF, SW480, IGROV-1, and OVCAR-8) after siRUVBL1 (si#1, si#2) or siRUVBL2 (si#1, si#2) transfection. (B) Increase in mTORC1 activity enhances sensitivity to RUVBL1/2 silencing–induced cell death. Cell death was measured 6 days after siRUVBL2 transfection to SW480 shControl and SW480 shTSC2 cells. (C) Reduction in mTOR activity prevents cell death induced by RUVBL1 or RUVBL2 knockdown. To reduce mTOR activity in T24 cells, rapamycin or Torin1 (100 nM) was pretreated to T24 cells 16 hours before siRUVBL1/2 transfection. Cell death was measured 4 days (T24) or 6 days (OVCAR8) after transfection. (D) The effect of RUVBL1/2 knockdown on TTT and PIKK family proteins. Protein levels of RUVBL1/2, TTT, and PIKK family members were examined 48 hours after knockdown of RUVBL1-#2 and RUVBL2-#1. (E) RUVBL2 is overexpressed in human cancer tissues. Various human cancer and corresponding normal tissues were stained with RUVBL2 (green) and phospho-S6 (red), and RUVBL2 expression levels were quantified using ImageJ. Representative picture of RUVBL2 (green) and phospho-S6 (red) staining are shown from esophagus cancer and corresponding normal tissue. (**P < 0.01, significant difference of expression between normal and cancer tissues). (F) mTORC1 activity (p-S6) and RUVBL2 expression show positive correlation in human cancer tissues. (Pearson correlation coefficient = 0.70, P < 0.001). (G) Cancer cells with high mTORC1 activity require RUVBL1/2 for survival. All data are presented as means ± SD. Significant differences were calculated by one-way ANOVA compared with DMSO-treated group for each siRNA treatment (***P < 0.001).