Bcl 2

CytoTox 96^® Non-Radioactive Cytotoxicity Assay kits (Promega), which are based on the colorimetric detection of the released enzyme lactate dehydrogenase (LDH), was used to determine specific cytotoxicity. Vγ9Vδ2 T cells (Effector, E) were co-cultured with LX-2 cells (Target, T), which were pretreated with N-BPs for 4 h at specific E/T ratios for 4 h (indicated in figure captions). In some cases, after 4 h co-cultured, the number and area of Vγ9Vδ2 T cells clusters were determined using IncuCyte (Essen BioScience) image analysis software. Three different locations per well were imaged with a 10 × objective lens. Clusters were defined as cell aggregates occupying an area at least 300 µm² and were displayed as the number and area of clusters per well. In some experiments, Transwell^® units (pore size 0.4 µm, Corning) were used to separate Vγ9Vδ2 T cells from LX-2 cells. In some cases, the neutralization antibodies anti-NKG2D (10 µg/mL, BD), anti-FasL (10 µg/mL, Biolegend), anti-TRAIL (10 µg/mL, Biolegend), anti-TCR γδ (10 µg/mL, Biolegend), and their relevant isotype controls, were individually added in the co-cultures to block the NKG2D-, FasL-, TRAIL-, and TCR γδ- mediated pathways. To block perforin and granzyme B pathways, the perforin inhibitor Concanamycin A (CMA, Selleck) (1 µg/mL) and granzyme B inactivator BCL-2 (1 µg/mL, R&D Systems) were used (15 (link)).

AR (Millipore Cat# 06-680, RRID:AB_310214), ARv7 (Precision antibody Cat# AG10008, RRID:AB_2631057), Bcl-2 (R&D Systems Cat# AF810, RRID:AB_355621; Cat# MAB8272, RRID:AB_10890789; Dako clone 124 Cat# M0887, RRID:AB_2064429), β4 integrin (Abcam, Cambridge, UK, #ab133682, RRID:AB_2923284, and 450-11 A, RRID:AB_396065, as in [24 (link)], β-actin (Abcam Cat# ab8227, RRID:AB_2305186), E-cadherin (GeneTex Cat# GTX100443, RRID:AB_10729586 and Abcam, #ab231303, RRID:AB_2923285), Cytokeratin (Agilent Cat# GA053, RRID:AB_2892089 and Santa Cruz Biotechnology Cat# sc-81,714, RRID:AB_2191222), Goat-anti-Mouse Alexa Fluor 546 (Thermo Fisher Scientific Cat# A-11,003, RRID:AB_2534071), Goat anti-Mouse IgG HRP (Bio-Rad Cat# 170–6516, RRID:AB_11125547), Goat-anti-Rabbit IgG Alexa Fluor 546 (Thermo Fisher Scientific Cat# A-11,010, RRID:AB_2534077), Goat-anti-Rabbit IgG HRP (SeraCare KPL Cat# 5220 − 0336, RRID:AB_2857917), IgG (Bethyl Cat# P120-101, RRID:AB_479829), JMJD3 (Abcam Cat# ab38113, RRID:AB_943898), H3K27me3 (Active Motif Cat# 39,155, RRID:AB_2561020), HSP90 (Cell Signaling Technology Cat# 4877, RRID:AB_2233307), UTX (Abcam Cat# ab36938, RRID:AB_883400).

After treatments, proteins were extracted from H9c2 cells or heart tissues lysed in a RIPA buffer containing 1% PMSF and 1% phosphatase inhibitor cocktail (Solarbio). The protein concentration was determined using a BCA protein assay kit (Solarbio). Equal protein extracts were analyzed by Western blotting, with primary antibodies against Bax, GRP78, GRP94, and PDIA6 (Abcam, Cambridge, U.K.); Bcl-2 (R&D Systems, Minneapolis, MN, U.S.A.); Caspase-12, ATF6, PERK, and p-PERK (Proteintech Group, Chicago, IL, U.S.A.); CHOP, cleaved Caspase-3, AMP-activated protein kinase (AMPK), and p-AMPK (Cell Signaling, Danvers, MA, U.S.A.); IRE1α, p-IRE1α, and peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) (Novus Biologicals, Littleton, CO, U.S.A.); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ImmunoWay, Plano, TX, U.S.A.). Immunoblots were detected by ECL (Millipore, Billerica, MA, U.S.A.) with anti-mouse or anti-rabbit IgG coupled to horseradish peroxidase as the secondary antibody (ImmunoWay). Gel images were captured using a Universal Hood II (Bio-Rad, Hercules, CA, U.S.A.) and quantitated with the ImageJ 1.51k software.

Snap-frozen LV tissue of the heart was obtained and lysed with RIPA solution (Beyotime Institute). Isolated protein (40 μg) was separated by 10% SDS-PAGE, transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, USA), and incubated at room temperature for 1 h. The membranes were then incubated with primary antibody overnight at 4°C. The following primary antibodies were used: Bax (1:1,000, Cell Signaling Technology, Boston, MA, USA) and Bcl-2 (1:1,000, R&D Systems, MN, USA). After four 5-min washes in TBST, the membranes were incubated with HRP-goat anti-rabbit (1:3,000, Elabscience, Wuhan, China) and HRP-goat anti-mouse (1:3,000, Elabscience, Wuhan, China) antibodies at room temperature for 1 h in the dark. After washes with TBST, the band intensities were analyzed using the Odyssey Imaging System (LICOR Biosciences, Lincoln, NE, USA).

Cells were lysed in the laemmli buffer (Biorad) containing 1X protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA, 78440). Cell lysates were run on a 4–12% SDS PAGE gel (Invitrogen NP0321BOX), the proteins were transferred to a PVDF membrane, and the membrane was blocked with 5% skim milk (VWR (Bridgeport, NJ, USA) 97063-958) in TBST (0.1% Tween20) and probed with target antibodies. Primary antibody incubations were performed overnight at 4 °C. Antibodies were used Rpb1 (CST 14958), p-Rpb1 (CST 4735), PARP (CST 9532; 1:500), cCP9 (CST 7237; 1:500), cCP3 (CST 9665; 1:500), Mcl-1 (CST 5453; 1:500), Bcl-xL (CST 2764; 1:500), Bcl-2 (CST 4223; 1:500), Noxa (Calbiochem (Burlington, MA, USA) OP180, clone 114C307; 1:500), β-actin (Sigma Aldrich A1978, clone AC15; 1:2000). The HRP linked secondary antibodies were from Santa Cruz Biotechnology Inc. The target protein was detected on the Azure (C300) imaging system. In selected cases, the protein capillary electrophoresis (Protein Simple (San Jose, CA, USA) SM-W004) was used. The following antibodies were applied Mcl-1 (CST 5453; 1:25), Bcl-xL (CST 2764; 1:25), Bcl-2 (R&D System, MN, USA, MAB827; 1:25), Noxa (Calbiochem OP180, clone 114C307; 1:25), and Vinculin (Abcam (Cambridge, MA, USA) ab129002, 1:500).

Total cellular RNA was isolated using TRIzol reagent (Gibco by Thermo Fisher, Waltham, MA, USA), and the concentration was determined using spectrophotometry. Both reverse transcription (RT) and PCR were performed using the Enhanced Avian HS RT-PCR Kit (Sigma-Aldrich, Burlington, MA, USA, MB-955). Specific primers (listed below) were used, and electrophoresis of the cDNA amplification product was conducted on an agarose gel. Gel images were scanned, and band intensity was quantified using a laser densitometer.
Primers:-

ACTB: s-5′-AGCGGGAAATCGTGCGTG-3′, a-5′-GGGTACATGGTGGTGCCTG-3′

-

Cox-2: s-5′-CCGGACAGGATTCTATGGAGA-3′, a-5′-CAATCATCAGGCGACAGGAGG-3′

-

Bax, Bcl-2, i-NOS, e-NOS: Purchased from R&D Systems Inc. (Minneapolis, MN, USA).

Supplementary Materials include phase-contrast microscope images, photos from a fluorescence microscope of von Willebrand factor, propidium iodide/annexin V preparations, DNA sticky ends in TUNEL technique, and actin cytoskeleton.