Fig. 1

Hyperosmolarity increases Lcn2-R expression in mIMCD3 cells and primary rat IMCD cells. a Expression levels of Lcn2-R mRNA by qPCR in mIMCD3 cells exposed to 300 (normosmolarity/-tonicity) or 600 mosmol/l (hyperosmolarity/-tonicity) for 24 - 72 h. Means ± SEM of 4-5 experiments are shown. The data obtained were normalized to the expression of the reference genes glyceraldehyde-3-phosphate dehydrogenase (Gapdh), β-actin (Actb), and β2-microglobulin (B2m). Statistical analysis compares the two osmotic conditions by unpaired t-test. n.s. = not significant. b Surface expression of Lcn2-R in mIMCD3 cells exposed to norm- or hyperosmotic media for 72 h. Lcn2-R was detected by live immunofluorescence microscopy of non-permeabilized cells using a Lcn2-R antibody directed against the extracellular N-terminus. Nuclei were counterstained with Hoechst 33342. Arrows indicate punctate staining along the plasma membranes of mIMCD3 cells. Statistical analysis shows means ± SEM of 4 experiments and compares the two osmotic conditions by unpaired t-test. a.u. = arbitrary units. c Immunoblotting of plasma membranes (PM) of mIMCD3 cells grown for 72 h in norm- or hyperosmotic media. Lcn2-R is expressed in PM at the expected molecular mass of ~62 kDa. The experiment is representative of three similar ones. d Histograms of Lcn2-R surface expression in rat primary IMCD cells exposed to 300 or 600 mosmol/l for 7 days. Lcn2-R was determined by flow cytometry using a Lcn2-R antibody directed against the extracellular N-terminus. The experiment is representative of three similar ones. e Expression of Lcn2-R in rat primary IMCD cells cultured in norm- or hyperosmotic media for a total of 7 days. Lcn2-R was detected by immunofluorescence microscopy of permeabilized cells using an Lcn2-R antibody directed against the N-terminus. Arrows indicate Lcn2-R staining along the plasma membranes of rat primary IMCD cells. Nuclei were counterstained with DAPI. The experiment is representative of three similar ones.