Characterizing TDP-43 Mutant in PC12 Cells

PC12 rat pheochromocytoma cells were maintained in DMEM supplemented with L-glutamine and 10% FBS at 37 °C in humidified condition of 5% CO₂/air. In some experiments, the cells were incubated with medium containing the chemical chaperone 4-PBA (TCI), the UPR activator tangeretin (TCI), pan-caspase inhibitors (zVAD-fmk from MBL and BocD-fmk from Abcam), an allosteric inhibitor of RIPK1 (necrostatin-1; Sigma), and JNK inhibitor (SP600125; LCL). 3 × FLAG-tagged human wt and Q343R mutant TDP-43 expression vectors were kindly gifted from Dr. Koji Yamanaka (Department of Neuroscience and Pathobiology, Nagoya University, Japan)52 (link). Plasmids were introduced into the cells using the FuGENE 6 (Roche) according to the manufacturer’s protocol. For RNAi-mediated gene silencing, the cells were transfected with target-specific or control scrambled siRNA using Lipofectamine RNAiMAX (Life Technologies). The following small interfering RNAs (siRNAs) were used: rat Hrs siRNA#1, 5′-CCAUCAAGAAGAAGGUCAAUGAUAA-3′; rat Hrs siRNA#2, 5′-UUAUCAUUGACCUUCUUCUUGAUGG-3′; and rat RIPK1 siRNA, 5′-GCGCUGAGUACAAUGAGGCUCUCUU-3′. Scrambled control siRNA (sc-37007) was purchased from Santa Cruz Biotechnology.