Anti total 4e bp1

The following primary Abs were used: rabbit polyclonal anti-phospho-Jak2 (Tyr^1007/1008), anti-phospho-Stat3 (Tyr⁷⁰⁵), anti-phospho-Stat5 (Tyr⁶⁹⁴), anti-phospho-Akt (Ser⁴⁷³), anti-phospho-eIF4E (Ser²⁰⁹), anti-phosp ho-4E-BP1 (Ser⁶⁵) used at 1/1000 dilution (Cell Signaling Technology), and mouse monoclonal anti-β-actin (1/5000; Sigma-Aldrich; St. Louis, MO) and anti-GAPDH (1/5000; Abcam, Cambridge, MA) antibodies. Goat anti-mouse EPOR (use at 0.2ug/ml) was purchased from R&D, and anti-total 4E-BP1, anti-total Jak2, anti-total eIF4E, and anti-total Akt were from cell Signaling. Secondary Abs included: anti-goat IgG-HRP (1/2500; Santa Cruz, Santa Cruz, CA), anti-rabbit and anti-mouse IgG-HRP (1/2000; Cell Signaling Technology).

Resveratrol (185-01721) and Hoechst 33342 (346-07951) were from Wako Pure Chemicals (Osaka, Japan). Food grade Resveratrol for mouse treatment was from ChromaDex (ASB-00018089-101, Irvine, CA). FITC-conjugated wheat germ agglutinin (WGA) lectin (W834), dihydroethidium (DHE) (D1168), MitoSOX Red (M36008), MitoTracker Red (MTR, M7512), and Lipofectamine RNAiMAX Transfection Reagent (13778-150) were from Thermo Fisher Scientific (Rockford, IL). The RNeasy Fibrous Tissue Mini Kit (74704) was from Qiagen (Valencia, CA). The GoScript Reverse Transcription System (A6010), GoTaq qPCR Master Mix (A600A), and ViaFect Transfection Reagent (E4982) were from Promega (Madison, WI). Antimycin A (A8674) and chloroquine (CQ) (C6628) were from Sigma-Aldrich (St. Louis, MO). Plasmid EGFP-LC3 Expression Vector was from Addgene (#11546). siRNAs against mouse Atg5 (SASI_Mm01_00089196), mouse Pink1 (SASI_Mm02_00331134), and MISSION siRNA Universal Negative Control (SIC-001) were from Sigma Genosys Japan (Ishikari, Japan). Antibodies used were as follows: anti-LC3AB (#12741), anti-phosho-Ser65-4EBP1 (#9451), anti-total 4EBP1 (#9452), and anti-ubiquitin (#3936) from Cell Signaling Technology (Beverly, MA), anti-p62 (GP62-C) from Progen (Heidelberg, Germany), and anti-GAPDH (G8795) and anti-α-tubulin (T5168) from Sigma-Aldrich.

Total protein from PLC/PRF/5, Huh7, sorafenib resistant Huh7, SNU423 and SNU449 were extracted with radio immunoprecipitation assay (RIPA) buffer. Cell protein lysates (20–30 μg) were separated on NuPAGE 4–12% Bis-Tris gels (Novex) electrophoretically and transferred to polyvinylidene difluoride membranes (Roche). Membranes were blocked for 1 h with 5% Bovine Serum Albumin in Tris-buffered saline containing 0.05% Tween 20 and incubated overnight with primary antibody. The following primary antibodies were used: anti-CD44 (Cell signaling, #3578), anti-vimentin (Abcam, ab92547), anti-total S6K (Cell signaling, # 2708), anti-phosphorylated S6K (Cell signaling, #9234), anti-total 4EBP-1 (Cell signaling, #9644), anti-phosphorylated 4EBP-1(Cell signaling, #2855) and anti-PARP (Cell signaling # 9532) Anti-GAPDH (Santa Cruz Biotechnology, sc-32233) and anti-β actin (Cell Signaling, 4970S) antibodies were used as loading controls. A secondary anti-rabbit (Cell Signaling) or anti-mouse immunoglobulin G (IgG) antibody peroxidase conjugate (GE Healthcare) was detected using ECL Western Blotting Analysis System (Amersham Biosciences).