Culturing and Treating Mouse Podocytes

The mouse podocyte cell line MPC5 used in our study was first established by Professor Peter Mundel et al. [26 (link)] in 1997, and it is widely used in many podocyte studies [26 (link), 27 (link)]. The culture method for MPC5 podocytes was similar to the originally described method. Briefly, podocyte proliferation occurred in a 33°C incubator using RPMI 1640 proliferation medium (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA), 100 U/ml penicillin-streptomycin (Gibco, USA) and 10 U/ml recombinant mouse interferon-γ (Sigma, USA). When the podocytes reached 60%-80% confluence, they were transferred to a 37°C incubator for differentiation for 10–14 days with RPMI 1640 medium in the absence of interferon-γ. The calpain inhibitor N-acetyl-Leu-Leu-Nle-CHO (ALLN) (Santa Cruz Biotechnology, USA) was dissolved in dimethyl sulfoxide (DMSO) (GeneChem, China), and the appropriate amount of DMSO was added to each control sample. PAN (Sigma, USA) was dissolved in water. Podocytes were treated with different concentrations of PAN (25, 50, 75, 100, or 125 μg/ml) for 24 h, and 75 μg/ml was selected as the appropriate concentration for subsequent studies, including migration assays, F-actin staining, and calpain inhibition experiments.