Fig. 6

Cortisol treatment of wild-type mouse retinal explants reinforces GR signalling and enhances glial homeostatic gene expression. A Experimental scheme of retinal explant cultures with persistent GR activation. B Left, Representative Western blots performed on whole retinal explant protein extracts when cortisol (500 ng/ml) was added twice daily. Right, Quantification of the total amount of GR and P-GR protein levels at 2 days of cortisol treatment. PDHB served as housekeeper to which the GR and P-GR were normalised to. Results of n=3 explants per treatment group are plotted as mean ± SEM. Unpaired t-test: *p<0.05. C Retinae from 6 control mice were cut into half. One half remained untreated and was kept under standard culture conditions. The other half was treated with cortisol (500 ng/ml, supplemented twice a day). Mass spectrometric profiling of explants after cortisol treatment over 48 h (n=6 biological replicates per treatment group) revealed 133 differentially expressed proteins. Using JASPER, ENCODE and CHEA databases, we identified those proteins whose genes are putative targets of GR and checked if those were then Müller cell-specific basing on our own RNA-seq data of purified retinal cell types. Eleven overlapping proteins were found amongst the up-regulated candidates, but none amongst the down-regulated upon cortisol treatment. D Pathway enrichment analysis of differentially expressed proteins from (D) using STRING. E Reanalysis of single cell RNA-seq data from Macosko et al. [64] confirmed Müller cell-specific expression of many of the 11 GR target genes found to be up-regulated upon cortisol treatment. F Protein quantification via mass spectrometry (n = 6 biological replicates per treatment group) of putative Müller cell-specific GR target genes as identified by filtering of data in (D). Unpaired t-test: *p<0.05. G–I Protein expression levels of select candidates are plotted on basis of quantitative mass spectrometric data collected from cortisol-treated retinal explants (n = 6 biological replicates per treatment group). Besides Nr3c1, its interaction partner RELA was chosen (H), and additionally Müller cell (I) and neuronal marker genes (J). GLUL, glutamine synthetase; KCNJ10, Kir4.1; RHO, rhodopsin; ARR3, cone arrestin; HOMER1, homer scaffold protein 1; CALB2; calretinin. J Sections from retinal explants were stained for the Müller cell marker glutamine synthetase (GLUL) and GR. K Quantitative real-time PCR (qPCR) of Müller cells purified form retinal explant cultures at 2 DIV was performed to determine mRNA levels of Glul and Gfap. Bars represent mean ± SEM (n = 3 for each condition). Scale bars, 20 µm.