Research Paper Volume 12, Issue 20 pp 20445—20456

Figure 4. Autophagy inhibition sensitizes CC-115 in RCC cells. 786-O cells were treated with CC-115 (5 μM) for 24h, expression of listed proteins in total cell lysates were shown (A); 786-O cells were pre-treated with 3-methyladenine (3-MA, 1 mM), chloroquine (Cq, 5 μM) or 0.5% DMSO vehicle control (“dmso”) for 1h, followed by CC-115 (5 μM) treatment for indicated time periods, cell viability and apoptosis were tested by CCK-8 assay (B) and ssDNA ELISA assay (C), respectively. Stable 786-O cells with Beclin-1 shRNA (“sh-Beclin1”), LC3 shRNA (“sh-LC3”) or scramble control shRNA (“sh-C”), were treated with CC-115 (1/5 μM) for indicated time periods, expression of listed proteins (D), cell viability (E) and apoptosis (F) were tested. Vector control or the “Beclin-1-OE” 786-O cells were treated with/without CC-115 (5 μM) for applied time; Listed proteins were tested (G); Cell viability (H) and apoptosis (I) were tested similarly. Primary human RCC cells (“RCC-1”), transfected with Beclin-1 siRNA-a (“siBeclin1-a”, Santa Cruz Biotech), Beclin-1 siRNA-b (“siBeclin1-b”, Cellular Signaling Tech) or scramble control siRNA (“siCtrl”) (200 nM each), were treated with/without CC-115 (5 μM) for applied time; Listed proteins were tested by Western blot assay (J); Cell viability (K) and apoptosis (L) were tested. “C” stands for parental control cells (H, I). Expression of listed proteins were quantified and normalized to the loading control (A, D, G, J). *P < 0.05 vs. untreated control group (“Ctrl”). ^# P < 0.05 vs. “dmso” cells (B, C). ^# P < 0.05 vs. “sh-C”/”si-C” cells (E, F, K, L). ^# P < 0.05 vs. Vector control cells (H, I).