Pf 4708671

FMK and BI-D1870 were purchased from Axon Medchem (Groningen, The Netherlands). PP2 was purchased from Cayman Chemical (Ann Arbor, MI, USA). PF-4708671 was purchased from Selleck Chemicals (Houston, TX, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (H₂DCFDA) and DAPI were purchased from Invitrogen (Carlsbad, CA, USA). TM, D-(+)-glucose and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were purchased from the following vendors: TXNIP (MBL International, Woburn, MA, USA); ChREBP (Novus Biologicals, Centennial, CO, USA); KDEL (GRP94, GRP78) (Enzo Life Sciences, Lörrach, Germany); CREB-2 (ATF4), GADD153 (CHOP) and Src (Santa Cruz, Santa Cruz, CA, USA); Akt, p-Akt, PARP, cleaved Caspase-3, p-Src, p-S6, S6, ACC, and FAS (Cell Signaling Technology; Danvers, MA, USA); Lamin B₁ (Invitrogen); α-tubulin (Sigma-Aldrich).

The human malignant prostate cancer cell lines PC-3, DU145; normal prostate benign hyperplasia (BPH-1) and normal fibroblast NIH3T3 cells, obtained from Sigma Aldrich (ECACC), were cultured in RPMI 1640 medium (Invitrogen Life Technologies, Carlsberg, CA, USA) supplemented with 10% FBS, 70 mg/L penicillin, 100 mg/L streptomycin, 6 mM HEPES and 2 mM L-glutamine (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO₂. The major inhibitors used in this study include AZD6244, MEK inhibitor; Rapamycin, mTOR inhibitor; CGP57380, Mnk1/Mnk2 inhibitor; LY294002, PI3K inhibitor; PF4708671, S6K inhibitor were purchased from Selleck chemicals, Houston, USA; BEZ235, AKT inhibitor and z-VAD, caspase inhibitor were purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA.

Small molecule inhibitors were purchased from Tocris Biosciences (R&D Systems): PF-4708671, U 73122, GSK2334470, IPA3, SL0101-1 and XMD 8-92 or from Selleck Chemicals LLC (TX, USA): PF-562271, Enzastaurin (LY317615), AUY922 (NVP-AUY922), 17-AAG (Geldanamycin), PF-04929113 (SNX-5422), AZD6244 (Selumetinib), AT7867, CHIR-98014, LY2228820, BIX 02188, AS703026, PH-797804, SP600125, NU7441. All inhibitors were prepaed in DMSO at 100 mM. Cells were treated with inhibitors prepared in culture media where the final concentration of DMSO was 2% v/v. Vehicle control treatments consisted of culture media containing 2% v/v DMSO. Six days after treatment, cell survival was measured in comparison to vehicle controls using the CellTiter 96® Assay as per manufacturer instructions (MTS assay, Promega Corporation, WI, USA). Data were analyzed in GraphPad Prism® version 5.00 for Windows (GraphPad Software, CA, USA) to measure the log₁₀ of IC50 for each drug. For combination assays, drugs were added simultaneously. Heatmaps for sensitivities (-log₁₀ of IC50) were prepared using D-chip Analyzer software [49 (link)].

HEK293, 293 T and DLD1 cells were obtained from American Type Culture Collection (ATCC), DLD1-PDK1^−/− and counterpart cells were kindly provided by Dr. Bert Vogelstein (Johns Hopkins University School of Medicine), and these cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. Cell transfection was performed using Lipofectamine and Plus reagents, as described previously^{44 (link)}. Packaging of lentiviral shRNA or cDNA expressing viruses and retroviral cDNA expressing viruses, as well as subsequent infection of various cell lines were performed according to the protocols described previously^{44 (link)}. Following viral infection, cells were maintained in the presence of puromycin (0.5 μg/ml).
Cell fractionations were performed with Cell Fractionation Kit (CST9038). Kinase inhibitors Mk2206 (Selleck S1078), Rapamycin (Selleck S1039), PP242 (Selleck S2218) and PF-4708671 (Selleck S2163) were used at the indicated doses. Growth factors including EGF (Sigma E9644) and insulin (Invitrogen 41400-045), were used at the indicated doses. PIP₃ beads (P-B00Ss) and label-free PIP₃ (P-3908) were purchased from Echelon Biosciences.

Ruxolitinib and PF-4708671 were purchased from Selleck Inc. (Shanghai, China). Gin A and antibodies against phospho-JAK2Y1007/Y1008 (sc-16566-R), β-actin (sc-47778), and GAPDH (sc-166574) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Antibodies against phospho-tyrosine (P-Y-1000, #8954), STAT3 (#9139), phospho-STAT3Y705 (#9145), JAK2 (#3230), AKT (#4691), phospho-AKTS473 (#4060), S6K1 (#2708), phospho-S6K1T389 (#9234), S6 (#2217), and phospho-S6S235/236 (#4858) were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-nucleoprotein (NP) polyclonal antibody was prepared by immunizing mice with purified recombinant NP protein. An anti-M1 monoclonal antibody was prepared in our lab. An anti-PB2 antibody (#GTX125926) was purchased from GeneTex Inc. (Irvine, CA, USA).

3-methyladenine (3-MA, 189490) and Bafilomycin A1 (BAFA1, 196000) were purchased from EMD Millipore (Darmstadt, Germany). Docetaxel and PF4708671 were from Selleck Chemicals (Houston, TX, USA). Lipofectamine 2000 reagent was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RIPA buffer was acquired from Cell Signaling Technology (Danvers, MA, USA), protease inhibitor and phosphatase inhibitor were from Abcam (Cambridge, MA, USA). Roche (Mannheim, Germany). Primary antibodies against LC-3 I/II, Beclin1, ATG3, ATG5, ATG7, mTOR, p-mTOR (Ser2448), ACC, p-ACC (Ser79), p-AMPK(Thr172), and AMPK were acquired from Cell Signaling Technology and HDAC3 was from Abcam (Cambridge, MA, USA). PVDF membrane was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). The siKLF5 #1, siKLF5 #2 siRNAs, siBeclin1 #1, siBeclin1 #2 siRNAs, siBcl2 #1, siBcl2 #2 siRNAs and the scrambled RNA, which used as shcontrol were obtained from RiboBio (Guangzhou, China). PLKO.1 lentiviral vectors encoding short hairpin RNA (shRNA) targeting non-specific control (NC) or human KLF5 (sh710: 5′-GGTTACCTTACAGTATCAACA-3′) were constructed by GenPharma (Shanghai, China).

Deferoxamine (DFO), CoCl₂, 2-methoxyestradiol (2-ME), N-acetyl-L-cysteine (NAC), MitoTEMPO, U0126, 5-aza-2’-deoxycytidine (5-aza-dC), Y294002, SB203580, Ro31-8220, rapamycin, BML-275, trichostatin A (TSA) and 2’,7’-dichlorofluorescein diacetate (DCF-DA) were obtained from Sigma-Aldrich (St Louis, MO. USA), and MitoPQ, MitoSOX-Red and 2‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (2‐NBD‐glucose) were from Abcam (Cambridge, UK), Thermo Fisher Scientific (Waltham, MA, USA) and Life Technologies (Carlsbad, CA, USA), respectively. AR-C155858 and SP600125 were supplied by Tocris Bioscience (Bristol, UK), and, syrosingopine was obtained from Extrasynthese SA (Lyon, France), respectively. TX-402 was a gift from Dr. H. Nagasawa, Gifu Pharmaceutical University. rapamycin was purchased from LC Laboratories (Woburn, MA, USA); BAY11-7082 was from AdipoGen Life Sciences (San Diego, CA, USA); PF-4708671 and KU-0063794 were from Selleck Chemicals LLC (Houston, TX, USA), and chaetocin was from Enzo Life Science (Farmingdale, NY, USA).