Fig 1: Cx43 HCs mediate cellular uptake of H2O2and excretion of oxidized glutathione. (A) Cells were pre-incubated with Cx43E2 antibody for 30 min and labeled with fluorescent Peroxide Sensor (Sigma-Aldrich) before the treatment with 0.3 mM H2O2 for various time periods. The intracellular H2O2 level was quantified by fluorescence intensity using NIH ImageJ software. The data are presented as the mean ± SEM. (n = 3). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared to non-H2O2 treated control (Two-way ANOVA). #, P < 0.05 compared to without Cx43E2 antibody control group (Two-way ANOVA). At least one microphotograph of fluorescence fields was taken under a 20X microscope (Keyence BZ-X710) using GFP filter. Scale bar: 50 μm. (B and C) HLE-B3 Cells pre-incubated with Cx43E2 antibody 30 min were treated with 0.3 mM H2O2 or UVB radiation for various time periods and intracellular and extracellular GSSG levels were tested using GSH/GSSG Ratio Detection Assay Kit (Fluorometric-Green, Abcam, ab138881). The data are presented as the mean ± SEM. (n = 3). *, P < 0.05; **, P < 0.01; ****, P < 0.0001 compared to non-H2O2 or UVB treated control (Two-way ANOVA). #, P < 0.05 compared to without Cx43E2 antibody control group (Two-way ANOVA). (D and E) HLE-B3 cells were transfected with Silencer™ Negative Control or Cx43 siRNA and then treated with 0.3 mM H2O2 or 63 mJ/cm2 UVB radiation. The intracellular H2O2 level was quantified by measuring fluorescence intensity, and the extracellular GSSG level were tested using GSH/GSSH Ratio Detection Assay Kit. The data are presented as the mean ± SEM. (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 compared to non-treated control (Two-way ANOVA). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig 2: OxPLs induce a metabolic shift to glutathione synthesis in macrophages. A. mRNA expression by qPCR measured in BMDMs treated with 1 μg/mL LTA (M1), 10 ng/mL IL4 (M2), or 10 μg/mL OxPAPC (Mox) for 4 h. Genes measured are Nos2 (iNOS; M1 marker), Arg1 (M2 marker), and Ho1 (Mox marker) (n ≥ 3). B. Metabolomics was performed on bone marrow-derived macrophages (BMDMs; harvested and cultured from C57BL/6 mouse hind legs) after 6 h treatment with M0 (vehicle, RPMI media), M1 (1 μg/mL LTA), M2 (10 ng/mL IL4), and Mox (10 μg/mL OxPAPC) stimuli (n = 5). Of a total of 512 metabolites of known identity, following normalization to protein concentration by Bradford assay, Welch's two-sample t-test was used to identify metabolites that differed significantly between experimental groups. A summary of the numbers of metabolites that achieved statistical significance (Welch's 2-sided t-test, p ≤ 0.05) either uniquely per individual treatment or commonly between treatments, represented in a Venn diagram (n = 5). C. Volcano plots produced using data from metabolomics of polarized BMDMs (see A) highlighting significantly (p ≤ 0.05) increased (red) or decreased (green) metabolites per group. Metabolites related to energy metabolism and redox homeostasis significantly changed in Mox (OxPAPC-treated) BMDMs are highlighted as red circles in each panel. Ornithine is highlighted in the M2 (IL4-treated) BMDMs as a positive control. D. Quantification of individual metabolites involved in the glutathione synthesis pathway in BMDMs represented by box and whisker plots. The highest and lowest bars represent the maximum distribution, while the upper, middle, and lower lines of the box represent the first quartile, median, and third quartile, respectively (n = 5). E. Glutathione synthesis pathway in Mox BMDMs (10 μg/mL OxPAPC, 6 h). #GSH (reduced glutathione) and GSSG (oxidized glutathione) levels were not measured. F. Intracellular reduced glutathione (GSH) levels in BMDMs treated for 6 or 24 h with vehicle (RPMI media) or 10 μg/mL OxPAPC, measured using the GSH/GSSG Ratio Detection Assay Kit (Abcam; ab138881) (n = 4). Data are expressed as mean ± SEM. Biological replicates indicated by (n). Statistical significance calculated by Welch's 2-sided t-test (*p ≤ 0.05; **p < 0.01; ***p < 0.001).
Supplier Page from Abcam for GSH/GSSG Ratio Detection Assay Kit (Fluorometric - Green)