On target plus control non targeting pool

HK-2 cells were purchased from AmericanType Culture Collection (Rockville, MD, USA). The cells were cultured in a medium consisting of DMEM/F12 (Gibco) supplemented with 10% FBS at intervals of 3-4 days to continuously passaged. HK-2 cells were cultured in DMEM/F12 medium for 16 h and cultured in the presence or absence of adipolin (300 ng/ml) for 1 h, followed by stimulation with Ang (1 μmol/L) or vehicle for 24 h. Knockdown of PPARα, HMGCS2, AdipoR1, or AdipoR2 was achieved by siRNA transduction at 10 nM with Lipofectamine RNAiMAX (Invitrogen) 24 h before experiments. Lipofectamine RNAiMAX and siRNAs were dissolved in Opti-MEM (Gibco). The ON-TARGETplus siRNA SMART pools targeting each gene were purchased from Horizon Discovery. Control cultures were transfected with an unrelated scrambled siRNA (ON-TARGET plus Control Non-Targeting Pool, Horizon Discovery).
Conditionally immortalized murine MPC-5 podocyte clonal cells (University of Bristol) were maintained in RPMI1640 medium containing 10% fetal bovine serum, 10 IU/ml recombinant murine interferon-γ (Wako) at 33°C for proliferating cells. To induce differentiation, MPC-5 cells were incubated at 5% CO2 at 37°C without interferon-γ for 14 days.