Mccoy 5a

PEG (M_n = 4000, Sigma Aldrich, St. Louis, MO, USA), ε-caprolactone (ε-CL, Alfa Aesar, Ward Hill, MA, USA), stannous octoate (Sn(Oct)₂, Aldrich), Cur (Sigma Aldrich, St. Louis, MO, USA, the purity more than 98%) and methyl thiazolyl tetrazolium (MTT, Sigma Aldrich, St. Louis, MO, USA) were performed without further purification. The extracellular domain of TRAIL (amino acids 114–281) was provided from Chengdu Huachuang Biotechnology Co. Ltd. HOOK^TM-Dye Labeling Kit (G-Biosciences, St Louis, MO, USA) was used to label TRAIL with fluorescent dyes. Anti-Ki67 antibody was purchased by Thermo Scientific, anti-DR4 and anti-DR5 antibodies were provided from Abcam, and caspase-3 and PARP antibody were obtained from Cell Signaling. Cell apoptosis was detected using TdT-mediated Dutp nick-end labeling (TUNEL) staining (in situ cell death detection kit, Madison, WI).
HCT116 colon cancer cells were incubated in 5% CO₂ atmosphere with McCoy 5 A (Gibco, Invitrogen, Grand Island, NY, USA) media supplemented with 10% fetal bovine serum (FBS, Gibco) at 37 °C. Trypsin-EDTA (0.25%) was applied for cell passaging.
Male nude mice were housed in groups of five, kept under pathogen-free conditions, fed with food and water ad libitum and acclimatized for a week. This study was granted by the Ethics Committee on Animal Experimentation of Sichuan University (Chengdu, China).

A498, ACHN, 786-O, HK-2, 769-P, OS-RC-2, Caki-1, Caki-2 were purchased from cell bank of Chinese Academy of Science, Shanghai. RCC4, RCC4/EV and RCC4/VHL were provided by Dr J.K. Cheng in SJTU-SM. There were no signs of mycoplasma contamination for all cell lines. RCC4, RCC4/EV and RCC4/VHL were cultured in DMEM medium (Invitrogen) supplemented with 10% FBS (Gibco BRL, Gaithersburg, MD, USA). A498, ACHN were cultured in MEM medium (Invitrogen) with 10% FBS, 786-O, HK-2, 769-P, OS-RC-2 were cultured in RPMI-1640 medium with 10% FBS, Caki-1and Caki-2 were cultured in McCoy’5A (Invitrogen) with 10% FBS. The cell lines were cultured in 5% CO₂/95% air in a humidified atmosphere at 37 °C. Hypoxic treatment was performed in a specially designed hypoxia incubator (Thermo Electron, Forma, MA, USA) with 1% O₂, 5% CO₂ and 93% N₂.

McCoy 5A, RPMI 1640, DMEM and DMEM/F-12 were purchased from Gibco (Grand Island, NY, USA). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Ipswich, MA, USA). The dual-luciferase reporter system and the empty psiCHECK-2 vector were purchased from Promega (Madison, WI, USA). Lipofectamine 2000 and Trizol reagent were obtained from Invitrogen (Carlsbad, CA, USA). TaqMan microRNA Reverse Transcription kits and TaqMan gene expression assays were from Applied Biosystems (Carlsbad, CA, USA). miR-103 precursors and inhibitors, scramble miRNA negative control were purchased from Ambion (Carlsbad). Lenti-miR-103 and Lenti-NC were from Genechem Biotech (Shanghai, China). Agomir-miR-103 and agomir-NC (miRNA mimics conjugated with cholesterol to make it more stable) were from Ribo Biotech (Guangzhou, China). Primary antibodies for DICER, PTEN and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA). Nucleotides were synthesized by TaKaRa (Shanghai, China). Transwell chambers were purchased from Costar (Cambridge, MA, USA).

ASCs were isolated from fat tissue obtained from three different donors as described previously [22 (link)] and maintained in Dulbecco's minimum essential medium enriched with sodium pyruvate and supplemented with 10% foetal bovine serum (FBS, Gibco Life Technologies), 100 U/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. The nonadherent cell population was removed after 48 h, and the adherent cell layer was washed twice with fresh medium; cells were then continuously cultured since their harvest until sixth passage. SaOs-2 (ATCC number: HTB-85) and MG-63 (ATCC number: CRL-1427) cells were, respectively, cultured in McCoy'5A (Gibco, Life Technologies) with 15% FBS (Benchmark, Gemini Bio-Products) and in Dulbecco's modified eagle's medium (DMEM, Gibco, Life Technologies) with 10% FBS. Both media were supplemented with 1% penicillin-streptomycin (MD Biomedicals, Thermo Fisher Scientific). Cells were always passaged at subconfluency to prevent contact inhibition and were kept under a humidified atmosphere of 5% CO2 in air, 37°C.

DMEM, McCoy’5A, RPMI 1640 were purchased from GIBCO company, and fetal bovine serum was purchased from PAN-Biotech; Restriction enzymes (PmeⅠ, SacⅡ), rTaq DNA polymerase, T4 DNA ligase purchase since NEB company; DNA Marker, PMD18-T and dNTPs were purchased from TaKaRa Company. Lipofectmine 3000 transfection reagent purchased from Invitrogen company; The plasmid extraction kit was purchased from OMEGA; DNA recovery kit and PCR product purification kit were purchased from Qiagen Company. DNA PCR primers were synthesized by Shanghai Sangon. Anti-human IgG4 Fc (HRP) antibodies, anti-mouse Bcl-2, VEGF-Trap and CD34 antibodies were purchased from Abcam. Anti-mouse β-actin, caspase-3, BAX, STAT3, AKT, p44/42MAPK(ERK1/2), phospho-STAT3 (named P-STAT3), phospho-AKT (named P-AKT) and phospho-p44/42MAPK(ERK1/2) (named P-ERK1/2) antibodies were purchased from cell signaling technology.