Lv con

For DEC1 overexpression, DLD-1 cells were seeded in a 6-well plate at the density of 1 × 10⁶ cells/well and then transfected by GenJet TM in vitro DNA Transfection Reagent II (SignaGen, Maryland, USA;Cat:SL100489). The transfection mixtures contained 1 μg Flag-CMV2 vector or Flag-DEC1 plasmid. The transfection medium was replaced with the regular medium 6 h thereafter. The transfected cells were used for subsequent experiments as described below.
For DEC1 knockdown experiment, DLD-1 cells were lentivirally transduced to LV-CON or LV-DEC1-RNAi (Genechem, Shanghai, China). After 48 h, cells were cultured in media containing 5 μg/ml of puromycin to select the stable cells for two weeks. For DEC1 sg-RNA/cas9 experiment, DLD-1 cells were transfected with vector control or lentivirus encoding sgDEC1/cas9 (Genechem, Shanghai, China). Later, cells were cultured in media containing puromycin (5 µg/mL) for two weeks. The cells were designated as sg-CON (control, polyclonal cells) and sg-DEC1 (sg-DEC1/cas9, polyclonal cells) in this study, respectively.

Dulbecco’s modified Eagle’s medium (DMEM) and penicillin/streptomycin were purchased from Hyclone (Logan, USA). Foetal bovine serum (FBS) was purchased from Gibco (South America). Anti-HIF1α monoclonal antibodies were purchased from CST (Boston, USA). Anti-VEGF monoclonal antibodies were purchased from RD (Minneapolis, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies, New Super ECL Assay kit, and anti-β-actin antibodies were purchased from KeyGEN BioTECH (Nanjing, China). Transwell chambers and Matrigel were purchased from Corning Life Sciences (8-μm pores, Tewksbury, MA, USA) and BD Biosciences (San Jose, CA, USA), respectively. The QRT-PCR kit was purchased from Takara (Shiga, Japan). The Cell Counting kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. The BCA Protein Assay Kit was purchased from Beyotime Biotechnology (Shanghai, China). Recombinant lentiviral expression vectors (LV-HIF1α, LV-Con, LV-ShHIF1α, and LV-Scramble), and polybrene were obtained from Genechem (Shanghai, China). BALB/c nude mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd (Shanghai, China).