However, such work is beyond the scope of this study. Taken together, these novel data demonstrate that the LXA4 counter-regulatory signal in renal fibrosis may be mediated, in part by let-7c induction and that downregulation of let-7c miRNA by TGF-��1 is important in driving newsletter subscribe fibrotic responses. Future work will investigate the specific role of let-7c in human renal fibrosis. Concise Methods Cell Culture A full list of abbreviations used in the manuscript is detailed in Supplemental Table 4. Immortalized human kidney epithelial cells (HK-2; ECACC, Porton Down, UK) were cultured at 37��C in a humidified atmosphere of 95% air/5% CO2, and maintained in DMEM-F12 (Sigma-Aldrich, Steinheim, Germany) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 ��g/ml streptomycin, 10 ng/ml endothelial growth factor, 36 ng/ml hydrocortisone, 3 pg/ml triiodothyronine, and 5 ��g/ml insulin, 5 ��g/ml transferrin, and 5 ng/ml selenium (ITS) solution (Sigma-Aldrich).

Primary human mesangial cells (Clonetics, Basel, Switzerland) were maintained in MCDB-131 medium (Gibco, Carlsbad, CA), supplemented with 10% (v/v) heat-inactivated FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 ��g/ml streptomycin. Normal rat kidney fibroblasts (NRK-49F cells) were maintained in DMEM, supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 ��g/ml streptomycin. After serum-starvation for 24 hours, cells were stimulated with vehicle (0.1% ethanol) or LXA4 (1.0��10?9 M; Merck, Calbiochem, Nottingham, UK) for 30 minutes and media was removed and replaced with media with or without TGF-��1 (10 ng/ml; PromoCell GmbH, Heidelberg, Germany) for 30 minutes to 48 hours.

HK-2 cells were incubated with pertussis toxin (50 ng/ml; List Biologic Laboratories Inc, Campbell, CA) for 16 hours before LXA4 or TGF-��1 stimulation. For all cell stimulations, three independent experiments were performed. Real-Time qPCR RNA extraction from HK-2 cells was performed using an RNeasy RNA extraction kit according to the manufacturer��s protocol (Qiagen, Crawley, UK). RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies, Berkshire, UK). Real-time TaqMan PCR was used to quantify relative gene expression levels of CDH1, CDH2, JAG1, TGF��R1, TGF��R2, COL1A1, COL1A2, THBS1, HES1, and FN1 using preoptimized gene expression assays (Applied Biosystems, Foster City, CA). 18s rRNA was used as an endogenous control for normalization. miRNA-enriched RNA was extracted AV-951 from HK-2 cells using a miRNeasy RNA extraction kit (Qiagen). Real-time TaqMan PCR of miRNAs was used to validate let-7c, let-7a, and miR-192 expression using miRNA expression assays (Applied Biosystems). RNU48 expression was selected as an endogenous control for normalization of target miRNAs.