293t human embryonic kidney cell line

H4, U87, and U118 human glioma cell lines and 293 T human embryonic kidney cell line were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified incubator of 5% CO₂. Cells were transiently transfected with polyetherimide for 293 T cells to detect the efficiency of sh-ARHGEF16 or sh-CKAP5 constructs (Additional file 2: Figure S1A-C), or with Lipofectamine 2000 for glioma cell lines according to the manufacturer’s instructions. LV systems were used to establish stable glioma cell lines overexpressing GLI2A or ARHGEF16 or to knock down GLI2 or ARHGEF16. Puromycin (0.5 μg/ml) was added to cultures to maintain stable overexpression in the cell lines.

The B16-F0 mouse melanoma cell line, HBEC3-KT human bronchial epithelial cell line, 4T1 mouse breast cancer cell line, 293T human embryonic kidney cell line, A375 human malignant melanoma cell line and B×PC-3 human pancreatic cancer cell line from American Type Culture Collection (ATCC, Manassas, VA, USA), and Huh7 human liver cancer cell line from Riken Bioresource Center, Japan, were cultured in Dulbecco’s modified Eagle medium (DMEM) culture medium supplemented with 10% foetal bovine serum (FBS), 100 U mL⁻¹ penicillin and 100 μg mL⁻¹ streptomycin. The cells were grown in a 5% carbon dioxide (CO₂) humidified incubator at 37 °C until 70–80% confluence.

Human umbilical vein endothelial cells (HuVECs) were purchased from Life Technologies and cultured in Medium 200 with low serum growth supplement (LSGS) (Carlsbad, CA). PC-3 and DU 145 human prostate cancer cell lines and the 293 T human embryonic kidney cell line were purchased from American Type Culture Collection (ATCC) (Manassas, VA), and propagated following ATCC’s instructions.
SiRNA against KLF5 (SiKLF5), developed in a previous study [64 (link)] with the sequence of AAGCUCACCUGAGGACUCATT, was used to knock down KLF5 in human cells at a concentration of 150 nM or as indicated in related figures. The control siRNA (siCtrl), AAUUCUCCGAACGUGUCACGUTT, was purchased from Thermo Fisher (Waltham, MA) used to bring the siRNA concentration to the same in concentration gradient RNAi assay.
PI3K inhibitors Wortmannin and LY294002 were purchased from Cell Signaling Technology (Danvers, MA), and dissolved in dimethyl sulfoxide (DMSO).
Antibodies used in this study included the following: anti-HIF1α and anti-PDGF-B from Novus Biologicals (Littleton, CO), anti-PDGF-D from Santa Cruz (Santa Cruz, CA), anti-phospho-AKT (S473), anti-AKT, anti-phospho-EGFR (Y1068), anti-EGFR, anti-phospho-ERK1/2 and anti-ERK1/2 from Cell Signaling Technology, anti-VEGF and anti-CD31 from Abcam (Cambridge, MA), and anti-β-actin from Sigma. The antibody against KLF5 has been described previously [23 (link)].