Vdac1

VEGF-A165 was obtained from Sigma-Aldrich (St. Louis, MO; V5765). SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas). Antibodies against the following proteins were used as the primary antibodies: CD31 (BD Pharmingen, San Jose, CA; 550274; Cell Signaling Technology, Danvers, MA;77699); β-actin and GAPDH (Santa Cruz Biotechnology, Dallas, Texas; sc-47778 and sc-137179); PCNA, Calreticulin, Cyt C, VDAC1, mitofusin-2 (MFN2), SRF, phosphorylated (p) SRF, VEGFR1, VEGFR2, pVEGFR2, and VEGFR3 (Cell Signaling Technology, Danvers, MA; 13110, 12238, 4280,4661, 9482, 5147,4261, 2893, 2478, 2479, 3408); IP3R1 (Abcam, Cambridge, MA; ab5804; ThermoFisher, Waltham, MA, PA1-901); calnexin (Abcam, Cambridge, MA; ab22595); FUNDC1 (Aviva Systems Biology, San Diego, CA; ARP53281), FUNDC1 (Novus Biologicals, LLC, Littleton CO, NBP1-81063; EMD Millipore Corporation, Temecula, CA, ABC506).

MTT, Cycloheximide (CHX), MG132 and Flag antibody were purchased from Sigma. Antibodies to Sirt3, COX IV, hexokinase II, VDAC1, Mcl-1, survivin and prohibitin were purchased from Cell Signaling Technologies. LC3 antibody was purchased from nanoTools. α-tubulin antibody was purchased from Santa Cruz. Atg12 antibody was purchased MBL. p62 antibody was purchased fromEngo Life Science. All cell culture media were purchased from Invitrogen. Western blot reagents were obtained from Pierce Biotechnology.

For the isolation of whole cell lysates, frozen LV tissue was rapidly homogenized in a buffer containing 50 mM Tris HCl, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% sodiumdeoxycholate and protease inhibitor cocktail (Sigma-Aldrich). Isolation of the membrane fraction was performed with the Mem-PER Plus Membrane Protein Extraction Kit (ThermoFisher Scientific). Proteins were quantified using the BCA Protein Assay (Pierce). 50 μg of protein were loaded on 10% or 12.5% SDS-PAGE gels. After electrophoresis, proteins were transferred to nitrocellulose membranes. Filters were blocked and incubated with antibodies against cytochrome oxidase I (COX I; ThermoFisher Scientific), Tfam (Santa Cruz), PGC-1alpha (EMD Millipore), Bcl-2, Bax, phospho-AMPK (Thr172), alpha-AMPK, phospho-p44/42 MAPK, p44/42 MAPK, phospho-Akt (Thr308 or Ser473), Akt, phospho-p38MAPK, p38MAPK, TOM20, VDAC1, GRK2 (all Cell Signaling), ADRB1 (Abcam) and GAPDH (Abcam). After incubation with a peroxidase-conjugated secondary antibody (1:10.000), blots were subjected to the enhanced chemiluminescent detection method with the Fusion FX7 imaging system (Peqlab).

293T, human NSCLC cell lines containing H1975, HCC827, and A549, and normal non-tumor cells HBE, these cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Following ATCC protocols, all cells were maintained in a humidified incubator (37°C, 5% CO₂) and underwent mycoplasma analysis every two months. Chemicals, incorporating SDS, NaCl, Tris base, DMSO, and lipofectamine™ 2000 (#11668019), were gained through Sigma-Aldrich (St. Louis, MO). Dioscin, cycloheximide (CHX) and MG132 were sourced from Selleck Chemicals (Houston, TX). The RPMI-1640 and DMEM culture media, along with Fetal Bovine Serum (FBS) and penicillin-streptomycin (P/S), were sourced from Invitrogen (Grand Island, NY) to support the cell culture process. Primary antibodies targeting ki67 (#ab16667, IHC: 1:300) and FbxL7 (#ab59149, IB: 1:1000) were obtained from Abcam (Cambridge, United Kingdom) for use in this study, cytochrome C (#11940, IB: 1:1000), survivin (#2808, IB: 1:1000, IP: 1:200, IHC: 1:200), ubiquitin (#3936, IB: 1:1000), β-actin (#3700, IB: 1:10000), α-tubulin (#3873, IB: 1:5000), VDAC1 (#4661, IB: 1:2000), Bax (#14796, IB: 1:1000), cleaved-caspase 3 (#9664, IB: 1:1000, IF: 1:400) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). We obtained the Flag-survivin construct from Origene (RC205935).

Human NSCLC cell HCC827 and the immortalized lung epithelial cells HBE and NL20 were obtained from American Type Culture Collection (ATCC, Manassas, VA). PC-9 cell line was a product of Sigma-Aldrich (St. Louis, MO). Both PC9 and HCC827 cells carry a Glu746-Ala750 deletion mutation in exon 19 of the EGFR [26 (link), 27 (link)]. The gefitinib resistant cell lines, HCC827-GR, and PC-9-GR were newly established in our laboratory by exposing HCC827 or PC-9 cells to gradually increasing concentrations of gefitinib (starting at 10 nM and ending with 400 nM) for approximately 5 months. All cells were maintained at the incubator according to the standard protocols and subjected to routinely checking for mycoplasma contamination. Antibodies against p-c-Met (#3077), c-Met (#8198), cleaved-PARP (#5625), p-Akt (#4060), Bax (#14796), Akt (#4691), VDAC1 (#4661), p-ERK1/2 (#4370), β-actin (#3700), ERK1/2 (#9102), α-tubulin (#2144), cleaved-caspase 3 (#9664), cytochrome c (#4280), and ubiquitin (#3936) were obtained from Cell Signaling Technology, Inc. (Beverly, MA). The natural compound Licochalcone A (>97%) was from Selleck Chemicals (Houston, TX). Lipofectamine 2000 transfection reagent for transient transfection was purchased from Thermo Fisher Scientific (Waltham, MA).

Antibodies that recognize ERK1/2, p-ERK1/2, RAF1, p-RAF1, AKT, p-AKT, MEK1/2, p-MEK1/2, GAPDH, COXIV, VDAC1, cleaved caspase-3, calnexin, PCNA, E-cadherin and vimentin were purchased from Cell Signaling Technology (Beverly, MA). Antibodies against β-actin, Myc, and Flag were obtained from Sigma-Aldrich (St Louis, MO). Antibodies that recognize cyclin D1 and CDK4 were purchased from Santa Cruz Biotechnology (Dallas, TX). ATAD3A antibody was purchased from Novus Biologicals (Abingdon, UK). CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) was obtained from Promega (Madison, WI). Texas-red phalloidin, D-luciferin bioluminescent substrate, and alamarBlue Cell Viability Reagent were purchased from ThermoFisher Scientific (Waltham, MA). The RAS inhibitor salirasib and the ERK1/2 inhibitor SCH772984 were obtained from SelleckChem (Houston, TX), and 4-nitroquinoline-1-oxide (4NQO) was purchased from Sigma-Aldrich (St. Louis, MO).