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Fig. 4 | Journal of Neuroinflammation

Fig. 4

From: The BET PROTAC inhibitor dBET6 protects against retinal degeneration and inhibits the cGAS-STING in response to light damage

Fig. 4

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

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