Lentivirus based shrnas

Cells were transfected with indicated plasmids or shRNAs using Lipofectamine 2000 (Thermo Fisher Scientific, China) according to the manufacturer's instructions. GSDMB and USP24 plasmids were purchased from Viogene Bioscience (China) and GENECHEM (China), respectively. Lentivirus-based shRNAs were obtained from Sigma-Aldrich (USA). 293T cells were transfected with shRNAs (shRNA: pVSV-G:pEQXV=1:1:2) using Lipofectamine^TM 2000 Transfection Reagent (Cat 11608019, Thermo Fisher Scientific, USA), culturing medium was refreshed 24 hours after transfection and then the cells were cultured for another 24 hours. After that the virus-containing medium was co-cultured with bladder cancer cell lines. 72 hours after puromycin screening, transfection-positive cells were harvested for MTS, Western blotting or xenografts analysis. Chemical reagents cryptotanshinone (Cat. No. S2285), EOAI3402143 (Cat. No. S6877), and MG132 (Cat. No. S2619) were purchased from Selleckchem.

AP nuclease expression was modulated using chemical as well as transgenic manipulations. AP nuclease activity was suppressed using APEX1 inhibitor (APE1 Inhibitor III; API3) (Axon Medchem LLC, Reston, VA), methoxyamine (MX) which inhibits AP nuclease activity by binding to aldehyde group of abasic site (Sigma Aldrich, Saint Louis, MO) and lentivirus based shRNAs targeting APEX1 and APEX2 (Sigma Aldrich, Saint Louis, MO). For overexpression, the plasmids carrying these genes under CMV promoter were used.