Sunitinib su11248 malate

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.

Sunitinib (SU11248) malate was purchased from Selleckchem (Houston, TX). For the development of sunitinib-resistant cells, parental cells were exposed to increasing concentrations of sunitinib. In brief, the RCC cell lines were treated with varying concentrations of sunitinib (0, 1, 2, 3, 4, 5, 7, 10, 20 and 50 μM). With the passage of time of every 4 days, it has been observed that A498 cells showed stable growth and eventually became confluent at a concentration of 2 µM, while 798-O cells showed stable growth to confluence at a concentration of 4 µM. It can be assumed that cells that grew to confluence had developed stable sunitinib-resistance after a period of 4 months or > 20 passages. The cells were constantly in sunitinib containing medium throughout the selection process.