Fig 1: Regulation of Kir4.1 and Kir4.2 gene expression in cultured RPE cells by pathogenic conditions. Real-time RT-PCR was carried out with RNA extracted from cells which were stimulated 2, 6, and 24 h. In (f), a 12-h stimulation was also examined on Kir4.2 gene expression. Relative mRNA levels are shown as folds of unstimulated control (1). (a) Effects of chemical hypoxia, induced by adding 150 µM CoCl2, on the Kir channel gene expression. (b) Effect of cell culture in a 0.2% O2-atmosphere. (c–f) Effects of oxidative stress (20 µM H2O2; (c)), hyperglycemia (25 mM glucose; (d)), extracellular hypoosmolarity (60% osmolarity; (e)), and hyperosmolarity (+100 mM NaCl; (f)). (g) Effects of hyperosmotic media prepared by adding 100 mM NaCl or 200 mM sucrose, or by coaddition of NaCl and sucrose, on the Kir4.2 gene expression. (h) Effect of tetracaine (1 mM) on the Kir4.2 mRNA level. (i) Dose-dependent effect of high NaCl on the Kir4.2 mRNA level. Different concentrations (10–100 mM) of NaCl were added to the culture media. Each bar shows data derived from 3–9 independent experiments with cells of different donors. Significant difference vs. unstimulated control: * p < 0.05.
Fig 2: Kir4.2 protein in cultured human RPE cells. (a) Immunolabeling of a cell culture with an anti-Kir4.2 antibody (red). Cell nuclei were stained with DAPI (blue). Bar, 20 µm. (b) Western blot analysis of Kir4.2 protein performed with lysates of cells from five donors. (c) Western blots of Kir4.2 protein performed with lysates of acutely isolated neuroretinas from two post-mortem donors (left side) and cytosolic and membrane protein extracts of cells from one donor (right side). (d) Western blot of Kir4.2 protein performed with lysate and membrane protein extract of cells from one donor. (a,c,d) The images show one example of three experiments carried out using cells of different donors. The following antibodies were used for western blotting: rabbit anti-human Kir4.2 (c) and mouse anti-human Kir4.2 (b,d).
Fig 3: Kir4.2 gene transcription and mRNA stability. The hyperosmotic and CoCl2-induced expression of the Kir4.2 gene in cultured RPE cells is mediated by stimulation of gene transcription; extracellular hyperosmolarity also induces an increase of Kir4.2 mRNA stability. The relative mRNA level is shown as fold of unstimulated control (1; (a)) and percent of the 0-h control (b). (a) The NaCl- and CoCl2-induced Kir4.2 gene expression was prevented by actinomycin D (ActD; 5 µg/mL) which blocks RNA polymerase II. The cells were cultured 12 h in the absence and presence of high (+100 mM) NaCl and 24 h in the absence and presence of CoCl2 (150 µM), respectively. (b) The stability of the Kir4.2 mRNA differed significantly (p < 0.05; ∆) between cells cultured in the presence of high NaCl compared to cells cultured under control conditions and stimulated by CoCl2, respectively. The cells were first stimulated for 12 h with high (+100 mM) NaCl, and 24 h with CoCl2 (150 µM), respectively. Thereafter, actinomycin D (5 µg/mL) was added, and total RNA was isolated at different times. Each bar represents data obtained in 3–7 independent experiments using cells of different donors. Significant difference vs. unstimulated control: * p < 0.05. Significant difference vs. NaCl and CoCl2 control, respectively: ● p < 0.05. Significant difference vs. 0-h control: ○ p < 0.05.
Fig 4: Functional characterization of KCNJ15/Kir4.2.a, KCNJ15 mRNA expression in ATB and HC individuals from multiple cohorts (Supplementary Table 36). Singapore: RNA-seq, this study; UK 2010, Gambia 2011: microarray. Red line, median. P value: two-tailed Mann–Whitney U test (case-control) and paired Wilcoxon signed-rank test (time course). b, 3D projection of a THP-1 monocyte infected with mcherry-BCG (red), stained for lysosomes (cyan) and KCNJ15/Kir4.2 (green). The images were acquired 24 h post infection by 3D-SIM. Scale bar, 2 µm. c, Confocal images of uninfected and mcherry-BCG-infected (blue pseudocolor) THP-1 monocytes stained for intracellular K+ (APG-4, green) and Kir4.2 (red), 24 h post infection. Scale bar, 2 µm. Data from 3 independent experiments. d, Left: patch-clamp recordings of THP-1 ControlOE and KCNJ15OE cells using a ramp protocol from -120 mV to 120 mV in 200 ms showing currents elicited at 4.5, 50 and 160 mM [K+]e. Right: average current amplitude recorded at -100 mV in 50 mM [K+]e is shown as a scatterplot, n = 7 and 13 cells for ControlOE and KCNJ15OE, respectively. Data from 2 independent experiments. e, Mycobacterial (BCG) growth in THP-1 monocytes; compiled data of n = 3 independent experiments, each in triplicate. f, Mtb growth after 24 h in scrambled control (–) and KCNJ15KD ( + ) THP-1 monocytes; compiled data of n = 3 independent experiments, each in triplicate. g, Mtb growth in KCNJ15-overexpressing (KCNJ15++) and control (empty vector) primary monocytes; n = 4 donors, 24 h post infection. CFU, colony-forming units. n = 2 independent experiments. h, Volcano plot of differential gene expression between KCNJ15++ and control cells. Apoptotic genes have been highlighted. P values from two-sided Wald test in DESeq2 package, FDR < 0.05. i–k, Flow cytometry data of ControlOE and KCNJ15OE THP-1 monocytes stained with Annexin V and PI. Data from 2 independent experiments, n = 6. l, Immunoblot of protein lysate of ControlOE and KCNJ15OE THP-1 cells with APAF1 and ß-actin. Data from 2 independent experiments. m, ControlOE and KCNJ15OE THP-1 cells stained for mROS using Mitosox red. Mean fluorescence intensity (MFI) data from n = 150 cells of either type. Top panel-representative images. Bottom panel-compiled data. P values in d, e and i–k were calculated using two-sided unpaired t-test, two-sided paired t-test in f, and two-sided Mann–Whitney U test in g and m. In d–f, mean ± s.e.m. Data in g and i–k are shown as box and whiskers, minimum to maximum. *P < = 0.05, **P < = 0.01, ***P < = 0.001, ****P < = 0.0001; NS, not statistically significant.Source data
Fig 5: Kir4.2 knockdown affects the proliferation and viability of cultured RPE cells. The expression levels of the Kir4.2 gene (a) and the VEGFA gene (b), the proliferation rate (c), and the viability (d) were investigated in cells transfected with Kir4.2 siRNA (siKir4.2) and nontargeted siRNA (siNon). The cells were cultured 24 h in the absence and presence of high (+100 mM) NaCl and CoCl2 (150 µM), respectively, as indicated by the panels of the bars. The data are shown as fold (1; (a,b)) and percent of unstimulated, nontransfected control cells (100%; (c,d)), respectively. Each bar represents data obtained in 4–10 independent experiments using cells of different donors. Significant difference vs. unstimulated, nontransfected control cells: * p < 0.05. Significant difference vs. NaCl and CoCl2 control, respectively, and transfection with siNon: ? p < 0.05. Significant difference between siKir4.2 and siNon: ? p < 0.05.
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