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Fig. 5 | Cell Communication and Signaling

Fig. 5

From: 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

Fig. 5

Silencing of β-catenin blunts alterations of Lcn2 and Lcn2-R expression induced by hyperosmolarity in mIMCD3 cells. a Following transfection with siRNA against β-catenin or control siRNA for 6 h, expression levels of β-catenin mRNA were determined by PCR in mIMCD3 cells exposed to norm- or hyperosmotic media for 24 h. The optical density (O.D.) of the signals was analyzed by densitometry using ImageJ software and β-catenin mRNA expression was normalized to the reference gene Gapdh. Means ± SEM of 11 experiments are shown. Statistical analysis compares the effects of control versus β-catenin siRNA by unpaired t-test. b Under the same experimental conditions and exposure to norm- or hyperosmotic media for 48 h, β-catenin protein expression was detected by immunoblotting. Expression was normalized by calculating the ratio of β-catenin to the loading control β-actin. Statistical analysis shows means ± SEM of 6 experiments and compares the effects of control versus β-catenin siRNA by unpaired t-test. c RT-PCR analysis of Lcn2-R and Lcn2 mRNA in mIMCD3 cells transfected with control or β-catenin siRNA as described in (a). Lcn2-R and Lcn2 mRNA expression was normalized to the reference gene Gapdh. The change of gene expression of Lcn2-R and Lcn2 induced by osmotic stress (Δ O.D. 600-300 mosmol/l for 24 h) without or with β-catenin siRNA was plotted as means ± SEM of 3-5 experiments. Statistical analysis compared β-catenin silencing to control siRNA by unpaired t-test. d Effect of β-catenin silencing on the expression of Lcn2-R protein in mIMCD3 cells. Lcn2-R protein was detected by immunofluorescence microscopy of permeabilized cells. Nuclei were counterstained with Hoechst 33342. Statistical analysis determines the effect of β-catenin silencing on the change of Lcn2-R expression induced by osmotic stress (Δ a.u. 600-300 mosmol/l for 48 h) by unpaired t-test. Means ± SEM of 5 different experiments are shown. a.u. = arbitrary units. e Effect of β-catenin silencing on the expression of non-glycosylated ~22-kDa precursor and glycosylated ~25-kDa mature Lcn2 in mIMCD3 cells exposed to norm- or hyperosmotic media for 30 h. Statistical analysis determines the effect of β-catenin silencing on the reduction of secretory Lcn2 protein in cells (ratio of 25 to 22-kDa Lcn2) induced by osmotic stress (Δ O.D. 600-300 mosmol/l for 30 h) using unpaired t-test. Means ± SEM of 5 different experiments are plotted. f Effect of β-catenin silencing on Lcn2 protein secretion into culture media. Lcn2 was detected by immunoblotting as a 25-kDa band (see Additional file 4: Figure S4C). Signals were analyzed by densitometry using ImageJ software and normalized to cell numbers. Statistical analysis determines the effect of β-catenin silencing on the decrease of secreted Lcn2 elicited by osmotic stress (Δ O.D. 600-300 mosmol/l for 30 h) by unpaired t-test. Means ± SEM of 5 experiments are plotted.

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