Sc 25792

The ethanol extract of the leaf of Garcinia subelliptica Merr. (catalog # FBM124-035) was purchased from the Korea Research Institute of Bioscience and Biotechnology (Daejeon, Korea). Doxorubicin (D1515), E-64 (E8640), pepstatin A (P4265), and hydroxychloroquine (H0915) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and rapamycin (#13346) was from Cayman (Ann Arbor, MI, USA). Antibodies against PARP (#9532), mTOR (#2972), phosphor-mTOR (S2448, #2971), ULK1 (#8054), phospho-ULK1(S757, #6888), and phospho-ULK (S317, #12753) were procured from Cell signaling (Danvers, MA, USA). Anti-p62/SQSTM1 antibody was obtained from Abcam (Cambridge, UK). Antibodies against β-actin (sc-477,778) and AMPKα (sc-25,792) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and LC3 (L7543) was from Sigma-Aldrich.

Primary antibodies to β-actin (SC-47778, 1:1000), CaMKK-ß (SC-50341, 1:1000), and AMPKα1/2 (SC-25792, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Primary antibodies to p-CaMKKβ (12818S, 1:500), mTOR (2983S, 1:1000), S6 ribosomal protein (2217S, 1:1000), and p-S6 ribosomal protein (2211S, 1:1000) were purchased from Cell Signaling (Danvers, MA). Anti-p-AMPKα1/2 (11183, 1:1000) and anti-p16 (41296, 1:1000) antibodies were from Signalway (College Park, MD, USA). Anti-LC3B (NB100-2220, 1:500) and anti-p21 (NBP2-29463, 1:500) antibodies were purchased from Novus (St. Louis, MO, USA). Anti-p-mTOR (ab63552) and anti-Ki-67 (ab15580) antibodies were purchased from Abcam (Cambridge, UK). Anti-mouse (31430), anti-rabbit (31460), anti-goat (31402), and Alexa Fluor 488 (A11008) immunoglobin G secondary antibodies (1:5000) were purchased from Invitrogen (Carlsbad, CA, USA). The anti-OR2H2 primary antibody (1:1000) was purchased from Antikoerper-online.de (Aachen, Germany). L-Cis diltiazem was from Abcam (Cambridge, UK). SQ22536 and thapsigargin were purchased from Enzo Life Science (Farmingdale, NY, USA) and U73122 was from Sigma-Aldrich (St. Louis, MO, USA). Aldehyde 13-13 was obtained from Henkel (Düsseldorf, Germany). A769662, an AMPK agonist, was purchased from Cayman (Ann Arbor, MI, USA). Rapamycin was obtained from LC Laboratories (Woburn, MA, USA).

Antibodies against ICAM-1 (SC-107), p-AMPK (SC-33524), AMPK (SC-25792), p-p38 (SC-166182), p38 (SC-271120) and β-actin (SC-47778) were all bought from Santa Cruz (Santa Cruz, CA, USA). All ON-TARGETplus siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Cell culture supplements were purchased from Invitrogen (Carlsbad, CA, USA). A Dual-Luciferase^® Reporter Assay System was bought from Promega (Madison, WI, USA). qPCR primers and probes, as well as the Taqman^® one-step PCR Master Mix, were supplied by Applied Biosystems (Foster City, CA, USA). All other chemicals not mentioned above were supplied by Sigma-Aldrich (St. Louis, MO, USA).

The primary antibodies to AMPK (1:1,000 dilution, sc-25792; Santa Cruz Biotechnology, Inc., TX, USA), p-AMPKα1/2 (Thr183/172) (1:1,000 dilution, sc-101630; Santa Cruz Biotechnology, Inc.), peroxisome proliferator-activated receptor-γ (PPAR-γ, 1:1,000 dilution, ab209350; Abcam, Cambridge, UK), CPT-1 (H40) (1:1,000 dilution, sc-98834; Santa Cruz Biotechnology, Inc.), GLUT-4 (1:500 dilution, bs-0384R; Bioss Antibodies, MA, USA), PGC-1α (1:500 dilution, bs-1832R; Bioss Antibodies), Bax (1:1,000 dilution, ab32503; Abcam), Bcl-2 (1:500 dilution, bs-0032R; Bioss Antibodies), caspase-3 (1:500 dilution, bs-2593R; Bioss Antibodies), and horseradish peroxidase (HRP) goat anti-rabbit (IgG) secondary antibody (1:5,000 dilution, BV-S8008; BIOVAL, CA, USA) were used. To document the loading controls, the membrane was reported with a primary antibody against anti-rabbit HRP secondary antibody. The signals were quantified by scanning densitometry and computer-assisted image analysis.

Expression levels of AMPK and LC3 protein were measured via Western blotting in the cytoplasm fraction from fresh liver tissues as previously described.^{(32 (link))} Protein concentrations were determined with a Pierce BCA assay (Thermo Fisher Scientific GmbH). Briefly, 30 µg of protein per sample was heated with loading buffer, denatured at 95°C for 5 min and separated by SDS-PAGE (BioRad, Munich, Germany). The fluorescence of the proteins was activated by UV exposure for 5 min before transferring the proteins onto a PVDF membrane (BioRad). Samples were blocked with skim milk dissolved in Tris-buffered saline plus 0.05% (v/v) Tween 20. Proteins (LC3, AMPK and pAMPK) were identified using respective primary antibodies (LC3: 1:500, NB100-2220, Novus Biologicals, Wiesbaden, Germany; AMPK: 1:100, sc-25792, Santa Cruz Biotechnology, Heidelberg, Germany; pAMPK (Thr172) 1:1,000, 2535, Cell Signaling, Frankfurt, Germany) and a secondary antibody (Santa Cruz Biotechnology). Protein bands were visualized with ECL reagents (Fisher Scientific, Schwerte, Germany) in a ChemiDoc XRS system (BioRad), and band intensities were calculated with Image Lab 5.0 Software (BioRad). Target protein expression was related to the total protein load per lane, assessed as PVDF membrane fluorescence.