Mccoy s 5a caki 1

The renal cancer cell lines (786-O, 769-P, ACHN, Caki-1, Caki-2) and human renal tubular epithelial cell line (HK-2) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 (786-O, 769-P); McCoy’s 5A (Caki-1, Caki-2); DMEM (ACHN) and DMEM/F12 (HK-2) (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, USA). PFK15 (PFK-015; Selleck, China), 2-Deoxy-d-glucose (2-DG; Selleck, China), Sunitinib (SU11248) malate (Sunitinib; Selleck, China), and dimethylsulfoxide (DMSO; Sigma–Aldrich, USA) were also used. Cells were transfected with control siRNA and siRNA-PFKFB3 using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, USA). QRT-PCR and Western blot assays were used to evaluate the efficiency of siRNA interference. Total RNA was isolated using Trizol (Invitrogen, Thermo Fisher Scientific, USA). HiScript III All-in-one RT SuperMix (Vazyme, China) was used for cDNA synthesis. qRT-PCR was performed with SYBR qPCR Master Mix (Vazyme, China) using StepOne Plus (Applied Biosystems, USA) and LightCycler 480 PCR instrument (Roche Diagnostics, Switzerland) according to the manufacturer’s instructions. The primers and siRNA Oligo used are listed in Additional file 1: Table S1.

The human proximal tubular epithelial (HK-2) cell line and human ccRCC cell lines (786-O, Caki-1, and RCC-JF) were purchased from Pricella. The cells were cultured in RPMI-1640 (786-O), McCoy's 5A (Caki-1) and DMEM (RCC-JF) media (Gibco, USA) supplemented with 10% fetal calf serum (FBS; Biolnd, Israel), 100 U/mL penicillin (Beyotime, China) and 100 μg/mL streptomycin (Beyotime, China) in a 37 °C incubator under 5% CO2.

RCC cell lines (786-O, 769-P, ACHN, Caki-1) and human renal tubular epithelial cell line (HK-2) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI 1640 (786-O, 769-P), McCoy's 5A (Caki-1), DMEM (ACHN) and DMEM/F12 (HK-2) (Gibco, Thermo Fisher Scientific, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, USA). The lentiviral vectors containing short hairpin RNAs targeting SPTBN1 (shSPTBN1) and negative control (shNC) were constructed and transfected.2-Deoxy-D-glucose (2-DG; Selleck, China), a glycolysis inhibitor, was applied to inhibit aerobic glycolysis. Cells were transfected with small interfering RNA (siRNA) and overexpression SPTBN1 plasmid (oeSPTBN1) using Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, USA).