Bcl xl

Human wt^xeno and HIP^xeno iPSCs were resuspended in 1 ml of diluted high-concentration Matrigel (Corning) including pro-survival cocktail (200 μM ZVAD and 100 nM BcL-xL (both Millipore)), 200 ng ml⁻¹ IGF-1 (PeproTech), 100 μM pinacidil and 200 nM cyclosporine A (both Sigma-Aldrich). Forty million cells were injected using 3-ml 20-gauge syringes into four different injection sites (10 million per injection site) on the back of rhesus macaques, two below the scapulae and two above the iliac crest on either side of the spine. At each cell implantation site, the skin was pinched to create a tent, and cells were injected very slowly. Mild pressure was applied to the puncture wounds for approximately 10 s to prevent cell leakage. Buprenorphine SR (0.2 mg kg⁻¹) and meloxicam SR (0.3 mg kg⁻¹) were given subcutaneously for analgesia, and animals were returned to home housing.

ABT263, WEHI‐539, A1210477, GW3965, LXR623, ABT199, and adenosine triphosphate (ATP) were obtained from Selleckchem. Low‐density lipoprotein (LDL) from human plasma was acquired from Thermo Fisher. l‐Aspartic acid sodium salt monohydrate was purchased from Sigma. Reagents were dissolved in dimethylsulfoxide (DMSO) (10 mM stock solution) and stored at −20°C. Final concentrations of DMSO were below 0.1% (v/v). The following antibodies were used: ABCA1 (Abcam ab18180; 1:25), Mcl‐1 (Cell Signaling Technology (CST) 5453SS; 1:500), BAK (CST 12105S; 1:500), Bcl‐2 (Abcam ab59348; 1:500), BIM (CST 2933S; 1:500), Bcl‐xL (CST 2764S; 1:500), Usp9X (CST 15751S; 1:1,000), Noxa (Millipore OP180; 1:500), β‐actin (Sigma‐Aldrich A1978, clone AC15; 1:2,000), ATF3 (CST 33593S; 1:500), ATF4 (CST 11815S; 1:500), Vinculin (Abcam ab129002; 1:200), AMPK (CST 5831S; 1:25), pAMPK (CST 2531S; 1:25), PAPRP (CST 9532S; 1:500), caspase‐9 (CST 9502S; 1:500), GRP78 (CST 3177S; 1:25), HA (CST 3724S; 1:500), LXRβ (Abcam ab56237; 1:25), SDHB (Abcam ab14714, 1:25), OXPHOS (Abcam ab110411; 1:500), and secondary goat anti‐mouse IgG‐HRP‐linked (SC2005) and secondary goat anti‐rabbit IgG‐HRP‐linked antibodies (SC2004) were purchased from Santa Cruz Biotechnology, Inc.

mRNA was extracted 1, 3, 9, and 24 hours after mEHT treatment of cultured tumor cells using RNeasy Mini Kit (#74104, Qiagen, Venlo, Netherlands). Complementary DNA synthesis was done with RevertAid First Standard cDNA Synthesis Kit (#K1622, Thermo Scientific, Waltham MA, USA). QPCR testing of RPLP0 (housekeeping gene), PUMA, BAX, BAK1, XIAP, BCL‐2, BCL‐XL, and P21 gene expression (Table 1; all primer pairs were purchased from Sigma‐Aldrich, St Luis, USA was performed with CFX Connect Real‐Time PCR Detection System (Bio Rad, California, USA) using the SsoAdvanced Universal SYBR Green Supermix (#1725271, Thermo Scientific)) according to the vendors instructions. The fold‐change of the genes of interest relative to RpLp0 was defined as 2^−ΔΔCT values.

Primary antibodies against the following were used: HIF-1α (mouse monoclonal, 1:1 500, MAB5382; Chemicon Inc, Billerica, MD); BNIP3 (mouse monoclonal, 1:3000, B7931; Sigma, St. Louis, MI); BCL-xL (rabbit polyclonal, SAB3500349; 1:1000, Sigma, St. Louis, MI); VEGF (rabbit polyclonal, 1:500, PB0084; Boster, Wuhan, China); GAPDH (mouse monoclonal, 1:10000, KC-5G4; KangCheng, Shanghai, China); and β-tubulin (mouse monoclonal, 1:1000, A06868; Boster, Wuhan, China). Horseradish peroxidase-labeled secondary antibodies were obtained from Zymed Laboratories Inc (San Francisco, CA). Western blot assays were performed as previously described [16 (link)].

Protein samples were prepared from the lung tissues using 1× cell lysis buffer (Cell Signalling Technology, USA). Equivalent amounts of proteins (10–20 µg) were used to assess protein expressions as described previously [11 (link),12 (link)]. Briefly, protein was denatured by boiling, separated by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE, 12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked with 8% non-fat milk/PBS-T (PBS with 0.5% Tween-20) for 2 h and respectively incubated with the following rabbit-derived primary antibodies: NF-κB, Bax, Bcl-xL, cleaved-Caspase-3 and β-actin (all 1:1000 dilution; Sigma Aldrich, St. Louis, MO, USA). After washing, the membranes were probed with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5000 dilution; Cell Signalling Technology, USA). Proteins of interest were visualised using the enhanced chemiluminescence kit (EMD Millipore, USA), and the band intensities were quantified by densitometry using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Proteins were extracted from lung tissues, and their concentrations were measured using the Bradford assay. Proteins were separated using a polyacrylamide gel followed by transfer to a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies including iNOS, HO-1, NF-κB, Bax, and Bcl-xL (Sigma-Aldrich, St. Louis, MO, USA) overnight at 4°C, followed by incubation with the appropriate secondary antibodies (Santa Cruz Biotechnology, Oregon, USA). An enhanced chemiluminescence reagent (Santa Cruz Biotechnology Oregon, USA) was then added to visualize the proteins.

PW06 (Figure 1) was generated by Dr. Jin-Cherng Lien (College of Pharmacy, China Medical University, Taichung, Taiwan). DAPI, propidium iodide (PI), and trypsin-EDTA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), L-glutamine, and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, California, USA). Primary antibodies against -Bid, -Bax, -p53, -Bcl-2, -Bcl-xL,-XIAP, -cytochrome c, -caspase-9, -caspase-3, -caspase-8, -PARP, -Fas, -Endo G, and peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Alternatively, primary antibodies against β-actin, Bcl-xL, Bak, and AIF were purchased from Sigma-Aldrich (St. Louis, MO, USA).