Total parp

HepG2 cells (ATCC, Manassas, VA) derived from a human hepatoblastoma^{37 (link)} and Huh7 cells derived from a well-differentiated HCC^{38 (link)} were cultured in minimum essential medium (ATCC) and Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA), respectively. Withaferin A (WA) was purchased from Calbiochem EMD Millipore (Billerica, MA). Antibodies for phospho-ERK, phospho-ELK1, phospho-RSK, phospho-p38, ERK, ELK1, RSK, DR5, Cleaved Caspase 3, Bax, cleaved-PARP, total-PARP, PCNA and β-actin were purchased from Cell-Signaling Technology (Danvers, MA). U0126 was procured from Sigma-Aldrich (St. Louis, MO).

Commercially available antibodies to HRP-conjugated p53, cyclin D1, CDK2, TIM23, LC3, p62, phospho-p53 (Ser392 and Ser46), p19-ARF, cMyc, Caspase 8, DRAM1, and LAMP2 were purchased from Santa Cruz Biotechnology, CA. Phospho-MDM2 (Ser166 and Ser186), total MDM2, cleaved PARP, total PARP, pro-caspase 9, Caspase 3, phospho-Drp1 (Ser616), Beclin1, phospho-Ulk1 (Ser757), phospho-AMPKα (Thr172), phospho-Beclin1 (Ser15), and phospho-mTOR (Ser2481) were procured from Cell Signaling Technology, MA. HRP-conjugated antibody to actin, Mdivi1, Z-VAD-fmk, chloroquine, and FH535 were purchased from Sigma-Aldrich, MO. LAPTM4B was purchased from Novus Biologicals, CO. Commercially available AZD5363, rapamycin, and mTOR inhibitor (AZD8055) were procured from Cayman Chemicals, MI.

Antibodies for p110α, p-85, AKT, p-AKT ^Thr308, p-AKT ^ser473, S6, p-S6 ^Ser235/236, cleaved PARP, total PARP, cyclin D1, p-MET (1234/1235) and anti-MAPK antibodies (ERK and p-ERK) were from Cell Signaling (Danvers, MA). Antibodies against total Met, p-MET (pY1349 and pY1003) and Alexa Fluor Phalloidin 594 were from Invitrogen (Grand Island, NY). PIP3 antibody was from MBL Co. Ltd (Japan). β-actin antibody was from Sigma (St. Louis, MO).
Recombinant human HGF was from R&D Systems (Minneapolis, MN). Wortmannin and LY294002 were from Cell Signaling. Crizotinib, GDC-0941, GDC-0980, ARQ 197 and NVP-BEZ235 were purchased from Selleck (Houston, TX). Stock solutions were prepared in DMSO and stored at −20°C till further use.

Cells were lysed using the mammalian protein extraction reagent RIPA (Beyotime, Beijing, China) supplemented with a protease inhibitor cocktail (Roche, Pleasanton, CA, USA), and phenylmethylsulfonyl fluoride (PMSF) (Roche). Approximately 50 μg protein extract was separated by 10 % SDS-PAGE, transferred to 0.22 μm nitrocellulose (NC) (Sigma), and incubated with specific antibodies. Autoradiograms were quantified by densitometry using Quantity One software (Bio-Rad, Brea, CA, USA). Rabbit anti-XIAP, cleaved caspase-3, total caspase-3, cleaved PARP, and total PARP were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH antibody was used as a control.

To confirm ADAR1 isoform specific KO and MDA5 KOs, CTRL A549s and various KOs cells were treated with 1000U/ml of universal type I IFN (PBL Assay Science) for 24 hours and lysed for western blot analysis. To confirm RIG-I KO, vector control and KO cells were infected with SeV for 16 hours and then lysed for western blot analysis. Whole cell lysates for western blot analysis was prepared using RIPA buffer (50 mM Tris pH 7.8, 150 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate, 1% Triton X-100) containing protease inhibitors and quantified by Bradford protein assay. Approximately 50–80 μg of total protein was loaded on to SDS gel. Antibodies used for western blot analysis: ADAR1 (Abcam 88574; Santa Cruz sc-271854), MDA5 (Alexis Biochemicals AT113), RIG-I ((1C3 clone) [82 (link)]), IRF3 (Santa Cruz sc-9082), V5 (Bio-Rad), cleaved PARP (Cell Signaling 5625), total PARP (Cell Signaling 9542), and Ku (#K2882 Sigma).

Antibodies (Ab) against Chk1, Chk1-pSer296, Chk1-pSer317, Chk1-pSer345, p21, beta-Actin, H2AX, H2AX-pSer139, total PARP, cleaved-PARP, total caspase-3, cleaved-caspase-3, c-Myc, Bcl-2, Mcl-1, STAT3-pSer727, and STAT3-pTyr705 were purchased from Cell Signaling Technology. Anti-NFκB Ab was from Santa Cruz Biotechnology. Anti-p53 Ab (DO-1) was a gift from Dr. Borivoj Vojtesek (Masaryk Memorial Cancer Institute, Brno). Anti-rabbit and anti-mouse secondary antibodies were purchased from DakoCytomation. Anti-Ki-67-PE Ab and appropriate isotype control were purchased from BioLegend.

19-HB was prepared from an aqueous extract of Chanpi and was prepared as a stock solution of 10 mM in DMSO. Western blot antibodies targeting Bcl-2, Bax, cleaved-caspase3, total PARP, cleaved-PARP, MMP2, MMP7, MMP9, c-Myc, N‐cadherin, β-catenin, Vimentin, Snail, Slug, and Cyclin D1 were purchased from Cell Signaling Technology (Beverly, MA, USA), and the antibody against α-Tubulin was bought from Proteintech (Rosemont, IL, USA).