Fig 2: Carbon ion radiation suppress BCL-x expression in photon resistant NPC cells. (A) Elispot analysis of apoptotic and necroptotic regulators in CNE-2 and CNE-2-RR cells at 48 hours in irradiated cells. (B) Expression levels of GM-CSF at 48 hours after carbon ion irradiation. Values presented are mean ± SEM from 3 independent measurements. *P=0.05, **P=0.01. And schematic diagram of necroptotic cascade induced by carbon ion beams in NPC cells. After repeated photon irradiation, NPC cells showed photon resistance and presented relatively more predisposed to necroptosis compared to the parental cells.

Fig 3: (A) Bax and Bcl-xL interaction. Binding of wild-type and S73D Bcl-xL to immobilized Bax. Bax was immobilized to anti-Bax antibody bound to Protein-G-Dynabeads and incubated with either wild-type (lanes 2, 4) or S73D (lanes 1, 3, 5) Bcl-xL. One incubation was performed without anti-Bax antibody (lane 1). Bax was obtained either from untreated cells (lanes 1–3) or from cells treated with tBid-expression adenovirus (lanes 4, 5). Bound proteins were eluted, electrophoresed by PAGE and probed for Bcl-xL. After localization of Bcl-xL, the membranes were probed for Bax. Asterisk, non-specific band from Protein-G-Dynabeads. (B) Bcl-xL on mitochondria and in cytoplasm. Bcl-xL immunoblot showing relative distribution of Bcl-xL in mitochondrial and cytoplasm fractions. Legend is the same as Figure 2c. For the cytoplasmic fraction, 10 µg protein was loaded, and for the mitochondrial fraction 1 µg protein was loaded. (C) Bcl-xL and Bax on mitochondria. Mitochondria were isolated from TKPTS cells and proteins extracted with 1% n-dodecyl-ß-D-maltoside. Proteins from mitochondria (lanes 1, 2) and cytoplasm (lanes 3, 4) were electrophoresed on BN-PAGE. Cells were transduced with either wild-type (lanes 1, 3) or S73D (lanes 2, 4) Bcl-xL adenovirus and incubated 2 days after transduction before harvesting. Western blots were probed for Bcl-xL (a) or Bax (b).

Fig 4: Endogenous Bcl-xL cleavage was associated with caspase 3 activation, and was downstream of Cdk2 activation. TKPTS cells were untreated and untransduced (lane 1) and treated with 25 µM cisplatin for 24 h (lanes 2, 3, 5, and 7). To inhibit Cdk2 activity, TKPTS cells were transduced with 100 MOI DN-Cdk2 24 h before cisplatin exposure (lanes 4 and 5) or treated with 9 µM purvalanol 1 h before cisplatin exposure (lanes 6 and 7). To inhibit caspase activity, 5 µM zVAD fmk was added 1 h before cisplatin exposure (lane 3). After 24 h of cisplatin treatment, cells were processed into (a) mitochondria/membrane fraction for western blot analysis of Bcl-xL and Tom20 (loading control), (b) supernatant fraction for western blot analysis of caspase 3 and ß-actin (loading control) and (c) DN-Cdk2. (d) Histone kinase assay was performed on untransduced TKPTS cells (lane a), TKPTS cells treated with purvalanol (lane b), and TKPTS cells transduced with DN-Cdk2 (lane c).

Fig 5: Expression of ADA-Bcl-xL and S73D-Bcl-xL resulted in perinuclear mitochondrial clustering. TKPTS cells transduced with WT-Bcl-xL, S73D-Bcl-xL, and ADA-Bcl-xL for 48 h were photographed using a Nikon Eclipse Ti-S fluorescent microscope. GFP expressing cells identified cells transduced with Bcl-xL. DAPI and Mitotracker Red CMXRos were used to stain nuclei and mitochondria, respectively. Asterisks represent dividing cells, which alters the intensity of GFP. White arrows indicate mitochondrial clustering. Merged pictures showed the perinuclear localization of clustered mitochondria in S73D-Bcl-xL- and ADA-Bcl-xL-expressing cells.