P p70s6k t421 s424

The primary antibodies and their dilution ratio in western blot analysis were as follows: p-mTOR-S2448 (1:1,000, Cell Signaling Technology); mTOR (1:1,000, Cell Signaling Technology); p-p70S6K-T389 (1:1,000, Cell Signaling Technology); p-p70S6K-T421/S424 (1:1,000, Cell Signaling Technology); p70S6K (1:1,000, Cell Signaling Technology); p-S6 Ribosomal Protein -S235/S236 (1:1,000, Cell Signaling Technology); RPS6 (1:1,000,Abcam); β-Actin (1:1,000, Tianjin Sungene Biotech Co., Ltd., China) and GAPDH (1:1,000, Good Here, China). DHA was purchased from Tokyo Chemical Industry (Cat #D3793, Japan). MK-2206 (AKT inhibitor, Cat #A10003) was purchased from Selleckchem. LY2584702 (p70S6K inhibitor, Cat #1082949–68–5) was purchased from Med Chem Express.

MPT0E028 and SAHA were synthesized by Professor Jing-Ping Liou. RPMI-1640 medium, FBS, penicillin, streptomycin, and all other tissue culture reagents were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), LY294002 and all of the other chemical reagents were purchased from Sigma Chemical (St. Louis, MO, USA). The following antibodies were used: caspase 8, caspase 9, p-Akt(T308), Akt, p-mTOR, mTOR, p-GSK3β, p-eIF4E, p-p70S6K(T421/S424), p-p70S6K(T389), HDAC1, HDAC2, HDAC4, acetyl-α-tubulin, BID, STAT2, STAT4, STAT5A, STAT6 (Cell Signaling Technologies, Boston, MA, USA); PARP, HDAC6, HRP-conjugated anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA); p-Akt (S473) (Epitomics, Burlingame, CA, USA); caspase 6, caspase 7, p53, STAT1 (BD Biosciences PharMingen, San Jose, CA, USA); caspase 3 (Imgenex, San diego, CA, USA); acetyl-histone H3, STAT5B (Upstate Biotechnology, Lake Placid, NY, USA); Actin (Chemicon, Billerica, MA, USA). Trizol reagent was from Invitrogen (Carlsbad, CA, USA). Random primer and M-MLRT were purchased from Promega (Madison, WI, USA). Pro-Teq was from Protech (Taipei, Taiwan).

Cells were seeded into 6‐well plates. Following overnight incubation, the cells were treated with LY294002, RAD001 or DMSO and harvested at 48 hours. Western blot analysis was performed as previously described.³² The membranes were exposed with an enhanced chemiluminescence reaction (ECL) kit, and the grey levels of the protein bands were analysed using the ImageJ 1.47v software. The sources of the primary antibodies applied were as follows: GAPDH (#2118), β‐tubulin(#2146), p‐PI3K S1981 (#4228), PI3K (#4249), p‐AKT S473(#4060), AKT(#4685), p‐mTOR S2448 (#5536), mTOR (#2983), p‐P70S6KT421/S424 (#9204), P70S6K(#2708), p‐S6 S235/236 (#4858) and S6 (#2217) were purchased from Cell Signaling Technologies; polyclonal rabbit anti‐12‐LOX was from Novus Biologicals (NBP2‐29941; Novus Biologicals; Centennial, CO, USA); VEGF antibody was obtained from Santa Cruz (sc‐7269; Santa Cruz; Dallas, TX, USA).