Dimethyl sulfoxide (dmso)

MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay was applied to detect cell proliferation ability. T24 and 5637 cells were seeded into 96-well culture plate 24 h prior to transfection with shRNA-ROR1-AS1 or shRNA-NC and miR-504 inhibitor or inhibitor NC. After 0, 24, 48 or 72 h transfection, 12 μl of MTT solution (TIANGEN Biotechnology, Beijing, China) at 5 mg/ml concentration was added to each well, and incubated for 3 h at 37°C. Then, the supernatants were discarded, and 120 μl of DMSO (TIANGEN Biotechnology) was used to solubilize the crystals for 20 min at 37°C. Data of absorbance were measured at a wavelength of 480 nm (with 590 nm as the reference wavelength) using a Synergy^™ HT Multi-Mode Microplate Reader (Biotek, Winooski, VT, USA) at the indicated time points.

MTT and DMSO were purchased from Tiangen Biochemical Technology Co., Ltd. (China). Glucose assay kit and glycogen staining kit and human insulin were purchased from Nanjing Jiancheng Co., Ltd. (China). High-glucose DMEM medium and phenol red-free 1640 medium and TRIzol and Lipofectamine 2000 were purchased from Thermo (USA). The PI3K inhibitor wortmannin S1952 was purchased from Biyuntian Biotechnology Company (China). Fetal bovine serum was purchased from Tianjin TBD Company (China). Trypsin was purchased from Promega (USA). cDNA first-strand synthesis kit and PCR mix and SYBR PCR mix were purchased from Takara (China). Protein extraction kit and BCA protein detection kit were purchased from Biyuntian Biotechnology Company (China).