Additional file 1: 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 S1. Hyperosmolarity inhibits proliferation and induces adaptive responses in mIMCD3 cells. (A) Proliferation of mIMCD3 cells cultured in media without (normosmolarity) or with 100 mmol/l urea and NaCl (hyperosmolarity) for 72 h. Measurements of MTT absorbance/optical density (O.D.) reflecting cell proliferation were performed at indicated time points. Means ± SEM of 5 experiments are shown. Statistical analysis compares the two osmotic conditions at each time point by unpaired t-test. (B) Expression levels of proliferation genes c-Myc (Myc) and cyclin D1 (Ccnd1) by qPCR in mIMCD3 cells exposed to norm- or hyperosmotic media for 72 h. The data obtained were normalized to the expression of the reference genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), and β2-microglobulin (B2m). Means ± SEM of 3-5 experiments are shown. Statistical analysis compares the two osmotic conditions by unpaired t-test. (C) Surface expression of Na+,K+-ATPase in mIMCD3 cells exposed to norm- or hyperosmotic media for 72 h. Immunofluorescence microscopy was performed in non-permeabilized cells. Nuclei were counterstained with Hoechst 33342. Images are representative of three individual experiments. (D) Expression of α1-subunit of Na+,K+-ATPase in homogenate (Ho) and plasma membranes (PM) of mIMCD3 cells grown for 72 h in norm- or hyperosmotic media. Expression was determined by immunoblotting. β-actin was used as a loading control. Image represents three independent experiments. (PDF 361 kb