Fig 1: dBET6 treatment inhibits STING expression in microglia/macrophages in response to LD. A, C Average expression of known microglia marker genes are used to identify the mouse (A) and human (C) retinal microglia cell cluster. Violin plots of genes previously reported to be enriched in microglia cell populations are plotted (cellmarker2.0, http://yikedaxue.slwshop.cn/). Mouse sc-RNA data are extracted from https://github.com/jiewwwang/Single-cell-retinal-regeneration, and human sc-RNA data can be accessed by GEO#: GSE137537. B, D Dot plot of cGAS-STING signaling genes and BRD4 in retinal microglia. E IHC shows indicated protein expression in mouse retina. Note STING-positive cells were localized to the GCL, IPL and INL in control mice. LD led to infiltration of IBA1-positive macrophages/microglia to the photoreceptors, and most of these infiltrating/reactive mononuclear phagocyte show STING staining (enlarged figure b). Administration of dBET6 reduced IBA1-positive cell in the photoreceptors and STING signal (enlarged figure c). Scale bar: 50 µm. D. IF analysis of retinal flat mounts. Note the evident overlapping of STING and IBA1 in both ramified (resting) and amoeboid-shaped (reactive) microglia/macrophages. Scale bar: 50 µm. E Quantification of STING IF intensity from the retinal flat mounts. For each group, six to ten regions were randomly selected in the whole mounts, the intensity of STING signal was measured by Image J. About 30 cells measured for Vehicle or LD + Vehicle, and ~ 60 cells measured for LD + dBET6. ****: p < 0.0001, One-way ANOVA, Tukey’s test, n = 3 retinas per group
Fig 2: dBET6 inhibits LD-induced microglia activation and gliosis. A IF analysis shows morphology of IBA1-labeled microglia in retinal flat mounts. B Quantification results of mean microglia processes and endpoints. For each group, six to ten regions were randomly selected in the whole mounts, total length of the branches and the total number of endpoints in the region were measured by Image J and divided by the IBA1-positive cell number. ns not significant, *: p < 0.05, ***: p < 0.0005, ****: p < 0.0001, n = 3 eyes per group, One-way ANOVA, Tukey’s test. For vehicle treatment, ~ 50 cells were measured, for for LD + vehicle or LD + dBET6, 130–200 cells were measured. C IF analysis shows IBA1-positive cells in mouse retina with indicated treatment. The macrophages/microglia were immune labeled by anti-IBA1 antibody. Scale bar: 50 µm. D Quantification results of IBA1-positive cells. For each treatment, five to eight regions of the whole retinas were randomly selected and the IBA1-positive cells were counted. For vehicle treatment, n = 3 eyes; for LD + vehicle or LD + dBET6, n = 4 eyes per group, respectively. *: p < 0.05, One-way ANOVA, Tukey’s test. E Representative WB analysis shows IBA1 protein levels with indicated treatment. Total retinal proteins were extracted and subject to WB analysis. F Quantification of WB analysis. **p < 0.01; ***p < 0.0005, n = 6–8 eyes per group, One-way ANOVA, Tukey’s test. G. IF analysis shows active macrophages/microglia labeled by anti-CD86. Arrows show CD86-positive cells infiltrating photoreceptor after LD. Arrow heads show CD86-positive cells in the GCL after LD. Scale bar: 100 µm. H Quantification results of CD86-positive cells. Two to three regions were randomly selected in the whole retina and the CD86-positive cells were counted. *: p < 0.05, **: p < 0.01, n = 3 eyes per group, One-way ANOVA, Tukey’s test. I WB analysis shows CD86 protein levels with indicated treatment. Total retinal proteins were extracted and subject to WB analysis. J Quantification of WB analysis. ns not significant, *: p < 0.05, n = 3 eyes per group, One-way ANOVA, Tukey’s test. K IF analysis shows GFAP signal in mouse retina with indicated treatment. Scale bar: 100 µm. L Quantification of GFAP fluorescence intensity. Four regions were randomly selected in the whole retina and the GFAP fluorescence intensity were measured. ***: p < 0.0005, n = 3 eyes for each treatment group, One-way ANOVA, Tukey’s test. M WB analysis shows GFAP protein levels with indicated treatment. Total retinal proteins were extracted and subject to WB analysis. N Quantification of WB analysis. ns not significant, *: p < 0.05, n = 3 eyes for each treatment group, One-way ANOVA, Tukey’s test
Fig 3: LD led to activation of cGAS-STING innate immunity in mouse retina. A–D Mice were expose with or without bright light and retinas were collected 48 h after exposure. Total RNAs or proteins were extracted and subjected to RNA-sequencing or WB analysis or IHC analysis, respectively. n = 4 for each group. A Gene Ontology (GO) analysis shows significant up- or down-regulated biological processes. Numbers indicate log10 and - log10 of the p. adjust. B Gene set enrichment analysis (GSEA) profiles demonstrate significant enrichment of gene sets associated with indicated pathway in light-exposed mouse retinas compared to control retinas. C Heat map shows increased cGAS-STING genes in LD group as compared with CTRL. p < 0.05. D WB analysis shows indicated protein level in mouse retinas. Right panels show the quantification results of the presented WB and a repeated WB not shown. *: p < 0.05, **: p < 0.01, ***: p < 0.001, n = 4 per group, unpaired t-test. E The DNA was labeled by anti-dsDNA antibody. Note that the cytosolic DNA seems to be engulfed by IBA1-positive microglia/macrophages in the ONL. Scale bar: 50 and 10 µm
Supplier Page from Proteintech Group Inc for IBA1 antibody