Ski 606

TRIzol RNA extraction reagent, SuperScript^™ III, Dulbecco's modified Eagle's medium, Opti-MEM medium, and caspase-3/7 green detection reagent were obtained from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was purchased from HyClone Laboratories (Logan, UT, USA). Metformin, rapamycin and chloroquine were purchased from Sigma (St. Louis, MO, USA). Compound C (AMPK inhibitor) was purchased from Calbiochem (San Diego, CA, USA). SKI-606 was purchased from Selleck Chemicals (Houston, TX, USA). Antibodies against CEBPD, cyclin D1, AMPK, p-AMPK and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against LC3B, p27, and p84 were purchased from GeneTex (Irvine, CA, USA). Antibodies against ATG3 and p62 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against HA, Src and p-Src were purchased from Covance (Berkeley, CA, USA). Antibodies against α-tubulin and mouse IgG were purchased from Sigma.

THP-1 cell lines were cultured in RPMI supplemented with 10% FCS, 100 μg ml⁻¹ of penicillin and 100 μg ml⁻¹ of streptomycin and 1 μg ml⁻¹ puromycin. ABT-199 was purchased from ChemieTek (Indianopolis, IN). Cantharidin, digoxigenin, proscardillin, wortmannin, SB203580, TOFA, IC 261 and vorinostat were all purchased from Sigma. ABT-737, GSK-J4, GSK-126, G007-LK, MM-102, SKI-606 and JNK-IN-8 were purchased from Sellekchem (Houston, TX). LB100 (HY-18597) was obtained from MedChemExpress (MonMouth Junction, NJ). After 4 days of Dox induction (or control without Dox), cells were plated in RPMI 20% foetal bovine serum at 2 × 10⁵ ml⁻¹ in 96-well plate with twofold dilutions of each drug performed in duplicate. At 72 h, cell viability was measured using a plate-reader after addition of 10% Presto Blue Cell Viability Reagent (ThermoFisher Scientific) at emission fluorescence 590 nm. IC₅₀ curves were calculated for each drug in the presence and absence of Dox using GraphPad Prism 6.0 (dose response inhibition) and the difference in IC₅₀ was plotted as a percentage of control (no dox). For ACACA validation, transduced THP-1 cells were induced with Dox for 3 days, then washed into low serum RPMI (0.5%) and cultured at low cell density for 7 days +/−TOFA before cell growth was measured with Presto Blue on a plate reader.

Deoxycholic acid (DCA) and JTE-013 were purchased from Sigma-Aldrich (St. Louis, MO). Z-Guggulsterone was purchased from Santa cruz Biotechnology (Santa cruz, CA). Phospherylated and total ERK, JNK, IκB antibodies and SP600125, SB 203580, U0126 were purchased from Cell Signaling Technologies (Beverly, MA). Vancomycin, QNZ and SKI-606 were obtained from Selleckchem (Houston, TX). Methoctramine was purchased from AdooQ Bioscience (Irvine, CA). SR 11302 was purchased from APExBIO (Houston, TX). C29 was obtained from MedChemExpress (Monmouth Junction, NJ). TLR2, Src antibodies were from Abcam and phosphotyrosine antibody (4G10) was from Millipore. ELISA Kits were obtained from eBioscience (San Diego, CA).