Livin

Western blot analysis was performed essentially as we described previously [13 (link)]. The membranes were probed with the following primary antibodies against: phospho-receptor-interacting protein kinase-1 (RIPK1) (Ser166) (#65746), cleaved caspase-3 (#9664), -7 (#8438), -8 (#9496), -9 (#7237), cleaved poly (ADP-ribose) polymerase (PARP) (#9541), cIAP-1 (#7065), cIAP-2 (#3130), XIAP (#2045), survivin (#2808), livin (#5471), STAT1 (#9712), phospho-STAT1 (#7649), STAT3 (#9139), JAK1 (#3332), JAK2 (#3229) (Cell Signaling Technology, Beverly, MA, USA), IFNγ receptor 1 (#AF673) (R & D Systems, Minneapolis, MN, USA), EGFR (#sc-03) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), vinculin (#V9131) and actin (#A4700) (Sigma. St. Louis, MO, USA). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) and detected with Bio-Rad Clarity Western ECL substrate (Hercules, CA, USA).

One hundred microliters RIPA lysis buffer (Beyotime, Zhejiang, China) containing 1 μL PMSF (Sigma, USA) was used to extract the cellular proteins. Concentrations of proteins were measured by BCA kit (Thermo Scientific, USA). Antibodies against Livin (Cell Signaling Technology, USA), H2A.X (Abcam, USA), H2A.XY142F (Abcam, USA), GAPDH (Santa Cruz, USA), ATG5 (Cell Signaling Technology, USA), ATG13 (Cell Signaling Technology, USA), ATG14 (Cell Signaling Technology, USA), LC3-I/II (Abcam, USA) and β-actin (Santa Cruz, USA) were used at 1:1000 dilution.

Western blot was performed according to the standard procedures. Primary antibodies against XIAP, cIAP1, cIAP2, livin, survivin were purchased from Cell Signaling Technology. E1A, caspase 9, caspase 8, caspase 3, PARP and TRAIL antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA), E1B 55KD was obtained from Oncogene (Cambridge, MA, USA) and GAPDH antibodies was from CoWin Bioscience (Beijing, China). All the secondary antibodies were purchased from Santa Cruz biotechnology.

Primary antibodies against Survivin (#2808), Apollon (#8745), Livin (#5471), Smac (#2954), cleaved caspase-8 (#9496), cleaved caspase-9 (#9505), cytochrome c (#4272), α-tubulin (#2144), COX IV (#4844), ubiquitin (P4D1, #3936) were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#sc-47724) were from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against NAIP (#ab25968), XIAP (#ab28151), c-IAP1 (#ab108361), c-IAP2 (#ab137393) were from Abcam (Cambridge, MA). Secondary antibodies for immunofluorescence were TRITC-conjugated donkey anti-mouse IgG and Alexafluor 488–conjugated goat anti-rabbit IgG, from Jackson ImmunoResearch Laboratories (West Grove, PA).

The Annexin V-FITC/PI kit was purchased from Invitrogen Corporation. Survivin, Ki67, livin, bcl-2, p53, VEGF, and MVD immunohistochemistry kits were purchased from Cell Signaling Technology Corporation. HepG2 cell line was provided by the Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. PHRE-Egr1-HSV-TK was constructed previously [1 (link)]. Hepatic cancer tissues of the control group, the radionuclide group, the MFH group, the radionuclide-gene group, and the radionuclide-gene-MFH group came from the previous study [1 (link)].

Total cell lysates were prepared using RIPA buffer (25 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) NP-40, 1% (w/v) sodium deoxycholate, 0.1% (w/v) SDS). Each protein sample (20–30 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes (Millipore, Bedford, MA). Antibodies against PAK2, Cyclin D3, CDK2, CDK4, CDK6, Cyclin D1, p18INK4C, p21Wafl/Cip1, p27Kip1, p53, Caspase 7, PARP, survivin, XIAP, Livin, Bad, phospho-Bad (S112), phospho-Bad (S136), and GAPDH were purchase from Cell Signaling Technology (Beverly, MA). Anti-Mcl-1, anti-Bax, HRP-conjugated goat anti-mouse IgG, and HRP-conjugated goat anti-rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin was purchased from Sigma. The labeled proteins were visualized with Immobilon Western Chemiluminesent HRP Substrate kit (Millipore) or Power Opti-ECL Western blotting Detection reagent (Bionote, Hwaseong, Korea) and the images were captured by ImageQuant LAS 4000 (GE healthcare, Buckinghamshire, UK). The protein band intensity on western blots was quantified and normalized to that of β-actin by the densitometry analysis using ImageJ software (http://rsb.info.nih.gov/ij/).

Lysates of A375 cells treated by the compound solution for 24 h were used to determine the change of IAP protein levels through western blotting. Primary rabbit antibodies against survivin, XIAP, cIAP1, cIAP2, livin and GAPDH were purchased from Cell Signaling Technology, Inc. (Danvers, MA) and used according to manufacture instructions as reported previously[19 (link)]. Protein lane intensities were quantified by ImageJ software (US National Institutes of Health, Bethesda, MD).