Insulin transferrin selenite supplement

Conditionally immortalized human podocytes were kindly provided by Dr. Moin A. Saleem (University of Bristol, Bristol, UK) and cultured and differentiated as described previously [43 (link)]. Briefly, podocytes were cultured with RPMI 1640 medium (Gibco, MD, catalog number 11875-093) supplemented with 10% fetal bovine serum (Gibco, MD, catalog number 10099-141) and insulin-transferrin-selenite supplement (Life Technologies, CA, catalog number 41400045). Podocytes were maintained in non-permissive conditions at 37 °C for 14 days to induce differentiation and then used for the experiments.

Conditionally immortalized human podocytes were provided by Dr. Moin Saleem, and cultured and differentiated as described previously56 (link)57 (link). Briefly, cells were cultured with RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaille, France) and insulin-transferrin-selenite supplement (LifeTechnologies, Carlsbad CA). Differentiated podocytes were transfected with pRc/RSV Flag MKK3, pRc/RSV Flag MKK3 (glu) or empty plasmid using Nucleofector Kit for primary Mammalian Epithelia cells (Lonza, Basel, Switzerland) as described previously57 (link). Transfected cells were incubated with vehicle or ANP, with the exchange of the medium containing ANP at every 24 h, and then harvested for RNA and protein analysis at 48 h. To confirm phosphorylation of MKP-1, differentiated podocytes were treated with ANP for 10 min and then harvested.