Pgph1 vector

The pEX-2 vector (Genepharma, China) was applied as a carrier for construction of AKIP1 overexpression [AKIP1(+)] plasmid and negative control (NC) overexpression [NC(+)] plasmid; the pGPH1 vector (Genepharma, China) was used as a carrier for construction of AKIP1 knockdown [AKIP1 (–)] plasmid and NC knockdown [NC(−) plasmid. The constructed plasmids were respectively transfected into AGS cells and MKN45 cells using Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA) following the instructions of the manufacturer, and the resulting cells were respectively named as AKIP1(+), NC(+), AKIP1(−), and NC(−) in each cell line. The cells without transfection were used as normal control. After transfection, all cells were incubated for 48 h under the hypoxia condition as described in the “Cell Culture and Hypoxia Treatment”. The Transwell assay, flow cytometry, sphere formation assay, RT-qPCR, and Western blot were performed.

The human colon cancer cell lines HCT116 and SW620 were obtained from the cell bank of the Chinese Academy of Sciences. All cells were maintained in Dulbeco’s Modified Eagle Medium (DMEM, Gibco, Grand Island, NY, US) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, US) and 1% penicillin-streptomycin solution (Gibco, Grand Island, NY, US) in a humidified atmosphere of 5% CO₂ at 37° C.
Specific small interfering RNAs (siRNAs) targeting GSPT1 (si-GSPT1) and negative control siRNA (si-NC) were acquired from GenePharma Co., Ltd. (Shanghai, China). Short hairpin RNA (shRNA) targeting GSPT1 and their sample control were designed by GenePharma (Shanghai, China). Oligos for expressing siRNA were then synthesized and inserted into the pGPH1 vector (GenePharma, Shanghai, China). For the RNA interference or GSPT1overexpression experiments, pGPH1 or pcDNA3.1-GSPT1 plasmids (1μg/well) and Lipofectamine 3000 (3μL/well) were mixed and applied to cells. For the siRNA experiments, 25pmol siRNA and sample control (NC) were mixed with 7.5μL Lipofectamine RNAiMAX reagent and applied for transfection. All transfected cells were cultured for 48h before the various assays were performed, unless indicated otherwise.

Short hairpin RNA (shRNA) targeting SOCS3 and its scramble control were designed by GenePharma (Shanghai, China). Oligos encoding the shRNA were then synthesized and inserted into the pGPH1 vector (GenePharma, Shanghai, China). MiR-25-3p mimics, inhibitors and their respective scramble control oligos were obtained from GenePharma (Shanghai, China). Cells were seeded onto 6-well plates. For transfection, the indicated plasmids and Lipofectamine 3000 (3 µL/well) were mixed and added to cells. For miRNA studies, 25 pmol of microRNA mimics/inhibitor or their scramble controls (NC) was mixed with 7.5 µL of Lipofectamine RNAiMAX reagent (Thermo Scientific, San Jose, CA, USA) and used for transfection. All the transfected cells were then cultured for 48 h before further assays unless indicated otherwise.