The Boston University mouse proximal tubular cell line (BUMPT) was originally provided by Drs. Wilfred Lieberthal and John Schwartz at Boston University School of Medicine (Boston, MA).[46 (link)
] Retrovirus PRDM16 (plasmid 15 504) and PRDM16 shRNA (plasmid 15 505) constructs were purchased from Addgene according to previously described.[47 (link)
] The PRDM16‐RFP stably expressed cell line was generated. Briefly, BUMPT cells were seeded in six‐well plates with DMEM containing 10% FBS and 1% penicillin‒streptomycin (Gibco, 15 140 163) at 37°C in 5% CO2. PB‐TRE‐PRDM16‐RFP (2 ug), PL623 (1 ug), and rtTA (1 ug) were transfected using Lipofectamine 2000 after plating for 24 h (Invitrogen) according to the manufacturer's instructions. After transfection for 24 h, 2 µg mL−1 puromycin was then added for 1 week, followed by selection of monoclonal cells that stably expressed PRDM16‐RFP.For experiment, BUMPT cells or the cell line stably expressing PRDM16‐RFP were treated by NG (5 mm D‐glucose), HG (30 mm D‐glucose), or mannitol (5 mm glucose+25 mm D‐mannitol) treatment for 24–48 h. BUMPT cells were transfected with AAV2 carrying TRPA1 knockdown and overexpression plasmids,negative control sequence (NC) and AAV2 vector used as a control, and then treated with NG or HG for 48 h.
] Retrovirus PRDM16 (plasmid 15 504) and PRDM16 shRNA (plasmid 15 505) constructs were purchased from Addgene according to previously described.[47 (link)
] The PRDM16‐RFP stably expressed cell line was generated. Briefly, BUMPT cells were seeded in six‐well plates with DMEM containing 10% FBS and 1% penicillin‒streptomycin (Gibco, 15 140 163) at 37°C in 5% CO2. PB‐TRE‐PRDM16‐RFP (2 ug), PL623 (1 ug), and rtTA (1 ug) were transfected using Lipofectamine 2000 after plating for 24 h (Invitrogen) according to the manufacturer's instructions. After transfection for 24 h, 2 µg mL−1 puromycin was then added for 1 week, followed by selection of monoclonal cells that stably expressed PRDM16‐RFP.For experiment, BUMPT cells or the cell line stably expressing PRDM16‐RFP were treated by NG (5 m