The murine kidney podocyte cell line, E11, was obtained from Cell Lines Service (Eppelheim, Germany). Undifferentiated E11 cells were plated in flasks precoated with collagen type I (Corning, Corning, NY, USA) and maintained in RPMI-1640 medium (Welgene Inc., Daegu, Republic of Korea), supplemented with 10% fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA), 1% penicillin–streptomycin (Welgene Inc.), and 10 U/mL recombinant mouse interferon (IFN)-γ (Miltenyi Biotec, Auburn, CA, USA) at 33 °C in an atmosphere of 5% CO2 and 95% air. To induce differentiation, the E11 cells were maintained at 37 °C without IFN-γ for 14 days.
To evaluate the effects of LPA, the medium of differentiated E11 cells was replaced with serum-free medium (SFM) containing 0.1% fatty acid-free bovine serum albumin (Sigma-Aldrich) and incubated for 18 h. The cells were treated with 5 μM LPA (Avanti Polar Lipids, Birmingham, AL, USA) in the presence or absence of 10 μM AM095 (Sigma-Aldrich).
To evaluate the effects of LPA, the medium of differentiated E11 cells was replaced with serum-free medium (SFM) containing 0.1% fatty acid-free bovine serum albumin (Sigma-Aldrich) and incubated for 18 h. The cells were treated with 5 μM LPA (Avanti Polar Lipids, Birmingham, AL, USA) in the presence or absence of 10 μM AM095 (Sigma-Aldrich).