Anti bcl 2

All cell culture and transfection reagents were from Invitrogen; and standard reagents were from Sigma or Fisher Scientific. Drugs were from: ABT-737 (Abbott Pharmaceuticals), ABT-263/PLX-4032/GSK-110212 (Selleck), zVAD-fmk (Calbiochem), Hoechst 33342 (Anaspec), and staurosporine/cisplatin/dacarbazine/vinblastine (Sigma). Antibodies (clone): anti-actin (C4), anti-A1 (FL-175), anti-BCL-2 (100), anti-CD31 (BD Pharmingen #550274) anti-MCL-1 (Rockland), anti-BAD (C7), anti-BIM (22 (link)–40), anti-PUMA (CT; Sigma), anti-SMAC (H177), anti-BID (C20), anti-cytochrome c (7H8.2C12), anti-BAK (G23), anti-BAX (6A7 for IP; N20 for western blot), anti-BCL-xL (H5 for IP; S18 for western blot), anti-GAPDH (9B3), anti-HSP60 (B-9), anti-p44/p42 MAPK ERK1/2 (137F5), and anti-phospho p44/42 MAPK ERK1/2 (197G2). Caspase-8 cleaved mouse BID (C8-BID) was from R&D Systems. Full-length BAX was made as described (24 (link)). Human BCL-xLΔC, MCL-1ΔC, and PUMAβ were made as described (17 (link)). The human BIM BH3 domain peptide (> 98% purity, Abgent) was resuspended in anhydrous DMSO in a N₂ environment, stored at −80°C, and thawed only once. All lipids for the LUVs studies were purchased from Avanti Polar Lipids. Statistical significance was evaluated by two tailed Student t test for p < 0.05.

Histological analysis of mouse organs was performed on 4 μm thick FFPE tissue sections, stained with Hematoxylin & Eosin (H&E) (Thermo Scientific)^{53 (link)}. The following primary antibodies and dilutions were used for immunohistochemical analysis: rabbit polyclonal anti-Bcl6 (1:300) (N3, Santa Cruz Biotechnology) and anti-Pax5 (1:400) (Neomarker); rabbit monoclonal anti-CD3 (1:800) (clone SP7, Neomarker) and anti-Ki67 (1:200) (clone SP6, Thermo Scientific); rat monoclonal anti-B220 (1:400) (clone RA3-6B2, BD Biosciences) and anti-CD138 (1:200) (clone 281-2, BD Biosciences); mouse monoclonal anti-BCL2 (1:200) (clone Bcl-2/100, BD Biosciences).

Primary antibodies used in this study were purchased from Cell Signaling Technology (Beverly, MA): anti-pPKA^Thr198/PKA, anti-pCREB^Ser133/CREB, anti-pSTAT3^Tyr705/STAT3, anti-pSrc^Ser17/Src, anti-pACC ^Ser79/ACC, anti-pAkt^Ser473/Akt, anti-pAMPK ^Thr172/AMPK, anti-p4EBP1^Thr37/46/4EBP1, anti-pmTOR^Ser2448/mTOR, anti-pP70S6K^Thr389/P70S6K, anti-EPAC-1, anti-BEATA2_AR, anti-FAK, anti-FASN, anti-GLUT4, anti-OCT3, anti-NOTCH, anti-PGC1, anti-pPRAS40^Thr246, anti-PRAS40, anti-pRAPTOR^Ser792, anti-RAPTOR, and anti-PI3K110α. Anti-BCL-2 was purchased from BD Bioscience (San Jose, CA). Anti-P27 was purchased from Thermo Fisher Scientific (Waltham, MA). Anti-rabbit immunoglobulin-horseradish peroxidase-conjugated secondary antibody, LumiGLO reagent with peroxide and cAMP assay kit were also purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-IGF1Rα, anti-HMGCR, anti-P21, anti-SCD1, and anti-SCEBP1 were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX); mouse anti-β-actin primary antibody was obtained from Sigma Aldrich (St. Louis, MO). The 1-methyl-1-nitrosourea (MNU) was obtained from Ash Stevens (Detroit, MI) and stored at −80°C prior to use. Metformin and buformin were obtained from Waco Pure Chemical Industries, (Waco, TX); phenformin was obtained from Sigma Aldrich (St. Louis, MO).

Protein expression was assessed as previously described (19 (link)). Antibodies used were: anti-MCL-1 (Rockland Immunochemical, PA), anti-human MCL-1, anti-PARP, anti-CHOP, and anti-BCL-X_L (Cell Signaling, MA). Anti-BCL-2 (BD Biosciences, CA), anti-NOXA and anti-PUMA (Sigma Aldrich, MO), and anti-Actin (Millipore, MA). Anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunochemical, ME. Immunoblots were acquired on a LI-COR Odyssey (LI-COR, NB) and densitometry assessed for all blots in replicate for statistical analysis (Sup. Fig. 7).