Additional file 4: of Tonicity inversely modulates lipocalin-2 (Lcn2/24p3/NGAL) receptor (SLC22A17) and Lcn2 expression via Wnt/β-catenin signaling in renal inner medullary collecting duct cells: implications for cell fate and bacterial infection

Figure S4. LPS induces Lcn2 expression and secretion and counteracts the effects of hyperosmolarity on Lcn2 and Lcn2-R expression in mIMCD3 cells. (A) Cells were cultured for 24 h in normosmotic medium with FBS, as described in the Methods and the medium was replaced by normosmotic medium without FBS ± LPS (5 μg/ml) for various time points. Medium was concentrated using Vivaspin 500 Centrifugal Concentrators (10 kDa MW cut-off) prior to immunoblotting. (B) Cells were cultured in normosmotic medium, as described in (A), prior to treatment with different concentrations of LPS for 18 h in the same medium without FBS. Medium was collected and Lcn2 secretion determined by immunoblotting, as described above. (C) mIMCD3 cells were cultured as described above and treated ± LPS (5 μg/ml) for 18 h in normosmotic medium without FBS prior to medium collection and measurement of Lcn2 secretion by immunoblotting. Cells were washed, scraped and homogenized by sonication in isosmotic sucrose buffer supplemented with protease inhibitors for immunoblotting. (D, E) mIMCD3 cells were exposed to norm- or hyperosmotic media for 24 h and treated ± LPS (5 mμg/ml) for additional 18 h in the same media without FBS prior to RNA isolation. RT-PCR shows mRNA expression for Lcn2 (D), Lcn2-R (E) and the reference gene Gapdh. The experiments are representative of at least three similar ones. (PDF 96 kb)