Pp242

In order to test the action of lomitapide in the control of cancer cell proliferation in the HCT116, HT29, and SW480 cell lines, the rate of cancer cell colony proliferation was examined by adding lomitapide to the wells in which cells were cultured. HCT116, HT29, and SW480 cells were inoculated in a 12-well plate at a density of 10⁵ cells/well and incubated at 37 °C for 24 h, and then the cells were treated with 0, 5 μM concentration of lomitapide. After lomitapide treatment, the plate was incubated at 5% CO₂ at 37 °C for 48 h. To measure colony formation of HT29^{FLAG-elF4E-GFP} stable cells, cells were seeded in a 12-well plate at a density of 1.25 × 10⁴ cells/well and incubated at 37 °C overnight, and then the cells were treated with a 2 μM concentration of lomitapide, rapamycin (Merck KGaA, Germany), and PP242 (Selleckchem, USA) for 96 h. Thereafter, 500 μl of crystal violet was added to each well, and cells were stained at room temperature for 10 min to analyze cell proliferation.

Autophagy inducer PP242 was purchased from Selleck chemicals (Houston, TX). Anti-CXCR4-APC (eBioscience, San Diego, CA, anti-VLA-4-APC (BD Bioscience, San Jose, CA) and anti-VCAM-1-APC (Biolegend, San Diego, CA) were used for FACs analysis. The following antibodies were used for Western blot: anti-Gli1 (Cell Signaling, Danvers, MA), anti-Gli2 (Santa Cruz, Dallas, TX), anti-Ptch1 (Santa Cruz, Dallas, TX), anti-FAK (AbCam, Cambridge, MA), anti-phospho-FAK (Cell Signaling, Danvers, MA), anti-paxillin (BD Biosciences, San Jose, CA), anti-phospho-paxillin (Cell Signaling, Danvers, MA), anti-SDF-1 (Santa Cruz, Dallas, TX), anti-IL-6 (R&D Systems, Minneapolis, MN), anti-LC3 (Novus Biologicals, Littleton, CO), and anti-GAPDH (Cell Signaling, Danvers, MA). Recombinant human SDF-1 (rhSDF-1) was purchased from Pepro Tech (Rocky Hill, NJ), and recombinant human IL-6 (rhIL-6) was purchased from R&D System (Minneapolis, MN). CXCR4 antagonist AMD3100, 2,7-dichlorodihydrofluorescein-diacetate (DCH-FDA, 50MG), ROS inhibitor N-acetyl-L-cysteine (NAC), FITC-labeled phalloidin, l-alpha-lysophosphatidylcholine, and autophagy inhibitor 3-MA were all purchased from Sigma-Aldrich (St Louis, MO).

The H2-K^b was amplified from C57BL/6 splenocytes and cloned into a pCDH-GFP vector by enzymes EcoRI and BamHI (NEB). The Acaca and Acly shRNAs were cloned into a pLKO.1-GFP vector. LPS, OVA_257-264, OVA_323-339, and OVA were purchased from InvivoGen. TOFA, C75, 2-DG, 2-NBDG, etomoxir, and BMS-303141 were purchased from Cayman Chemical. Rapamycin, KU0063794, PP242, and BPTES were from Selleck. Mitotracker and BODIPY were purchased from Invitrogen. UK5099, 25-HC, TMR-dextran, oligomycin, fluoro-carbonyl cyanide phenylhydrazone (FCCP), rotenone, and antimycin A were from Sigma. Antibodies for TSC1 (#4906), TSC2 (#4308), c-Myc (#5605), c-Fos (#2250), p-S6 (#4858), S6 (#2317), IRF1 (#8478), IRF5 (#4950), ACC1 (#3662), H3K9ac (#9649), p-ACLY (#4331), ACLY (#4332), p-IκBα (#2859), p-IKKα/β (#2697), p65 (#8242), and CREB (#9197) were from Cell Signaling Technology. Antibodies against H3K27ac (ab4729), H3K27me3 (ab6002), H3K9me3 (ab8898), and IKKα/β (ab178870) were purchased from Abcam. Antibody for IκBα (SC371) was from Santa Cruz Biotechnology. Antibody against GAPDH (AP0063) was from Bioworld.

HT-p21 cells, derived from HT1080 human fibrosarcoma cells, in which p21 expression can be turned on and off using IPTG (isopropyl-thio-galactosidase) were described [80 (link), 81 (link), 62 (link)]. HT-p21 cells were cultured in DMEM/10% FC2 serum (HyClone FetaClone II; HyClone Laboratories, Inc, Logan, UT), Immortalized WI38t human fibroblasts, described previously [111 (link)], and SKBR3, breast adenocarcinoma cell line (ATCC, Manassas, VA), were maintained in DMEM/10% FBS. Rapamycin was purchase from LC Laboratories (Woburn, MA). Torin 1 and PP242 were from Selleckchem (Houston, TX). Stock solutions were prepared in DMSO.

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.