Parp 1

Whole-cell extracts were prepared from cells using RIPA lysis buffer. Western blotting analysis was performed using anti-RIPK1 (BD Biosciences, San Jose, CA, USA), RIPK3, p21 (Santa Cruz), cIAP1/2 (R&D Systems, Minneapolis, MN, USA), PARP-1 (BD Biosciences) and cleaved PARP-1 (Cell Signaling Technology, Danvers, MA, USA) antibodies. Anti-HSP90 (BD Biosciences) and β-actin (Prosci, Poway, CA, USA) antibodies were used as a loading control.

The study of the expression of the main proteins involved in the extrinsic apoptotic pathway was carried out using Western blot analysis as previously described [19 (link),20 (link),21 (link)]. Briefly, cells (5 × 10⁶) were lysed at 4 °C with 100 µL of a buffer containing 1% Triton X-100 and protease and phosphatase inhibitors. Then, lysated cells were separated by 12% SDS-PAGE, transferred to PVDF membranes, and blocked with TBS-T buffer (10 mM Tris/HCl, pH 8.0, 0.12 M NaCl, 0.1% Tween-20, 0.05% sodium azide) containing 5% skimmed milk. PVDF membranes were incubated with mAbs against caspase-8 (BD Biosciences), caspase-3 (Cell Signaling, Danvers, MA; USA), Bid (BD Biosciences), PARP-1 (BD biosciences), caspase-9 (MBL, Woburn, MA; USA), cFLIP (clone NF6, Enzo, Farmingdale, NY, USA), Mcl-1 (Santa Cruz Biotech, Dallas, TX), Bcl-xL (Cell Signaling), or XIAP (BD Biosciences) in TBS-T containing 2% skimmed milk. Anti-β-actin mAb (Sigma, Saint Louis, MO; USA) was used as protein loading control. Pierce ECL Western Blotting Substrate (when used horseradish peroxidase-labeled secondary antibody, Life Technologies, Carlsbad, CA, USA) or the CDP-Star substrate (when used phosphatase alkaline-labeled secondary antibody, Merck, Darmstadt, Germany) were used to display the proteins.

After treatments, cells were collected and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 250 mM NaCl, and 0.1% Triton, and completed with protease (ThermoScientific, A32953) and phosphatase inhibitors (ThermoScientific, 88667). Total protein extracts were fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose filter, and subjected to immunoblot assay. Following primary antibodies were used: H3K9Ac (Millipore, 07–352); H3K56Ac (Abcam, ab76307); SIRT6 (Novus Biologicals, NBP1-30101); LC3B (Sigma-Aldrich, L7543); total AMP-activated protein kinase (AMPK; Cell Signaling, 2532) and phospho AMPK (Thr172; Cell Signaling, 2535); total (Calbiochem, ST1521) and phospho ULK1 (Ser555 and Ser757; Cell Signaling, 5869 and 6888 respectively); total mTOR (Cell Signaling, 4517) and phospho mTOR (ser2448; Cell Signaling, 2971); Beclin-1 (Cell Signaling, 3738); ATG5 (Cell Signaling, 2630); PARP1 (BD Pharmingen, 551025); β-actin (Sigma, A5441); HSP72/73 (Calbiochem, 386032); and H3 (Abcam, ab1791). Following secondary antibodies were used: Goat anti-mouse or anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase conjugated antibodies (Biorad, 1706516 and 1706515 respectively). Densitometry was performed using ImageJ software.

Immunoblots were performed according to [11 (link)]. For each membrane, a housekeeping protein is depicted (HSP90, GAPDH, vinculin), which is always shown underneath the detected proteins of interest. Antibodies were: BCL-XL (#ab32370), GAPDH (#ab128915), WT1 (#ab89901) from Abcam, Cambridge, U.K.; RAF1 (#sc-133), HSP90 (#sc-13119), vinculin (#sc-736), ɣH2AX (#sc-101696) from Santa Cruz, Heidelberg, Germany; cleaved caspase-3 (#cs9661), p-Tyr202/Tyr204-ERK1/ERK2 (#cs9101), ERK1/ERK2 (#cs9102) from Cell Signaling, Leiden, Netherlands; p-Tyr694-STAT5 (#MA5-14973) from Thermo Fisher, Braunschweig, Germany; STAT5 (#610192) from BD Bioscience, Heidelberg, Germany; and PARP1 (#556362) from Pharmingen, Heidelberg, Germany. The protein ladders used were the prestained Scientific^TM PageRuler^TM (#26617) and the PageRuler^TM Plus (#26620) from Thermo Fisher, Braunschweig, Germany.

The whole-cell lysates were resolved by SDS–PAGE, transferred to a polyvinylidene fluoride membrane and probed with primary antibodies against FLAG-M2 (1:2000; Sigma), MYC (1:2000; made in our laboratory), ORF45 (1:500; made in our laboratory), GFP (1:500; Santa Cruz Biotechnology), PARP-1 (1:1000, BD Biosciences) or α-tubulin (1:2000; Sigma). A goat anti-rabbit or goat anti-mouse immunoglobulin G antibody conjugated with horseradish peroxide (a secondary antibody; Santa Cruz Biotechnology) was detected using ECL plus Western blotting detection reagents (ELPIS), and the signals were analyzed on LAS-4000, a chemiluminescent image analyzer (Fujifilm).