Fig. 6
LPS reverts hyperosmolarity-induced changes of Lcn2-R/Lcn2 expression in mIMCD3 cells and decreases viability of CHO-K1 cells stably overexpressing hLCN2-R. mIMCD3 cells were exposed to norm- or hyperosmotic media for 24 h and ± LPS for additional 18 h. a RT-PCR analysis of Lcn2 mRNA, which was normalized to the reference gene Gapdh. Lcn2 gene expression, was plotted as means ± SEM of 4 experiments. Statistical analysis determines the effect of LPS by unpaired t-test. n.s. = not significant. b Effect of LPS (5 μg/ml for 18 h) on the cellular expression of 22-kDa precursor, 25-kDa mature Lcn2, and secretion of 25-kDa Lcn-2 in mIMCD3 cells. The experiment is representative of three similar ones. c Lcn2-R mRNA expression was determined by RT-PCR and normalized to the reference gene Gapdh. Means ± SEM of 6 experiments are shown. Statistical analysis compares LPS to control by unpaired t-test. n.s. = not significant. d Effect of LPS on the expression of Lcn2-R in mIMCD3 cells exposed to norm- or hyperosmotic media for 24 h and ± LPS (5 μg/ml) for additional 18 h. Lcn2-R protein was detected by immunofluorescence microscopy of permeabilized cells. Nuclei were counterstained with Hoechst 33342. Lcn2-R protein expression at 300 and 600 mosmol/l ± LPS was plotted as means ± SEM of 4 experiments. Statistical analysis determines the effect of LPS by unpaired t-test. a.u. = arbitrary units. e Both, CHO-K1 cell clones stably transfected with pcDNA3.1 or human lipocalin-2 receptor isoform B (hLCN2-R), when grown in hyperosmotic media secrete similar amounts of Lcn2 upon treatment with LPS (5 μg/ml for 48 h). Secretion of Lcn2 was determined by immunoblotting. The optical density (O.D.) of Lcn2 signals was quantified by densitometry using ImageJ software. Means ± SEM of 4 experiments are plotted. f Cell viability of both clones stably transfected with pcDNA3.1 or hLCN2-R was measured by MTT assay. Data show means ± SEM of 5-6 experiments with 6 replicates per experiment. Statistical analysis compares conditions ± hLCN2-R using unpaired t-test; n.s. = not significant.