Rpmi 1640 medium

The human kidney carcinoma cell line, 786-O, and the human embryonic kidney cell line, 293T, were purchased from American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 medium (Genepharma, Shanghai, China) supplemented with 10% fetal bovine serum (Invitrogen) in 5% CO₂ at 37°C. The primary antibodies anti-TGFBR1 and anti-JunB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and primary antibody anti-Actin was purchased from Beyotime Institute of Biotechnology (Jiangsu, China). Secondary antibodies anti-rabbit IgG and anti-mouse IgG were purchased from Beyotime and Santa Cruz, respectively.

Human glioblastoma cell lines U87, U251 and A172 were obtained from American Type Culture Collection (ATCC, American). Normal control cell (normal human astrocytes, NHA) was purchased from Sciencell Research Laboratories (American). RPMI-1640 medium supplement with 10% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin was used to routinely culture the cells at 37°C in 5% CO₂.
The miR-1225-5p mimic/inhibitor and negative control were purchased from Shanghai GenePharma Co., Ltd (Shanghai, China), which were used to upregulate/downregulate the expression of miR-1225-5p. For overexpression/knockdown of FNDC3B, we also synthesized si-con (5′-CGAACUCACUGGUCUGACC-3′), si-FNDC3B (5′-GAUGGAAAUUCUGAAGCGAAUCAGT-3′), pcDNA3.1-FNDC3B and corresponding control vectors from Shanghai GenePharma Co., Ltd (Shanghai, China). Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) was used for transfection, following the manufacturer’s protocol.

The human ccRCC cell line (786-O) was purchased from American Type Culture Collection (ATCC). All cells were maintained in RPMI 1640 medium (Genepharma, Shanghai, China) with 10% fetal bovine serum (Invitrogen) at 37 °C with 5% CO₂ [4 (link)]. The primary antibodies anti-E2F1, anti-GAPDH and the second antibodies anti-rabbit or anti-mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The powdery inhibitor of miR-29a-3p and negative control was bought from Genepharma (Shanghai, China). For E2F1 over expression vector, the E2F1 coding sequences were amplified and inserted into the pcDNA-3.1 vector (Invitrogen) using the XhoI and EcoRI restriction sites [39 (link)].