Fig. 3: ASP4132 activates programmed necrosis cascade in NSCLC cells.
From: AMPK activation by ASP4132 inhibits non-small cell lung cancer cell growth
Primary pNSCLC-1 cells were pretreated with the caspase-3 inhibitor z-DEVD-fmk (50 μM, 1 h pretreatment) or the pan caspase inhibitor z-VAD-fmk (50 μM, 1 h pretreatment), followed by ASP4132 (1 μM) treatment and cultured for applied time periods, cell apoptosis (apoptotic nuclei ratio, A), viability (CCK-8 OD, B) and cell death (Trypan blue positive cell ratio, C) were tested. Primary NSCLC cells, pNSCLC-1/-2, were treated with ASP4132 (1 μM) or the vehicle control, cells were further cultured for applied time periods, mitochondrial CyPD-p53-ANT1 association (Mito-IP, D), mitochondrial depolarization (by measuring JC-1 green monomers intensity, E, I) and cell necrosis (by measuring medium LDH contents, F, J) were tested. Expression of CyPD and Tubulin in stable pNSCLC-1 cells with CyPD shRNA lentiviral particles (sh-CyPD) or cyclosporin A (CsA, 5 μM, 24 h) treatment was shown (F). Control cells were treated with vehicle control plus scramble control shRNA lentiviral particles (“shC+DMSO”) (G); Cells were further treated with ASP4132 (1 μM) and cultured for 72 h, cell viability (CCK-8 OD) and cell death (Trypan blue positive cell ratio) were tested (H). Expression of listed proteins was quantified and normalized to the loading control (D, G). “Veh” stands for the vehicle control (Saline). Data were presented as mean ± standard deviation (SD, n = 5). *p < 0.05 vs. “Veh” cells. #p < 0.05 vs. “DMSO (0.2%)” pretreatment (A–C). #p < 0.05 vs. “shC + DMSO” cells (H). The experiments were repeated five times with similar results detected. Scale bar = 100 μm (E).
