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Additional file 1: Figure S1. Visual confirmation that glucocorticoid agonists enhanced retinal stem and progenitor yield and was not due to artifacts. Images from the Celigo imaging cytometer showing 96-well plate wells at the end of a 7-day growth assay. Wells were treated with the indicated glucocorticoid agonist compounds. The nuclear channel, the GFP channel and merge demonstrate the ability to differentiate Hoechst and actin-GFP double-positive objects from debris and other artifacts that appear only in the nuclear channel. Visually, it is apparent dexamethasone and prednisolone have greater signal than the other conditions. Red arrows indicate artifacts that fluoresce in the blue nuclear channel that do not fluoresce in the GFP channel. 4x magnification. Figure S2. Representative staining of cell death marker ethidium homodimer (EthD-1) and thymidine analog EdU. (A-B) EthD-1 labeling at Day 2 in cells treated with (A) 0.1% DMSO, or (B) 1μM Dexamethasone. (C-D) EdU labeling at Day 4 in cells treated with (A) 0.1% DMSO, or (B) 1μM Dexamethasone. Figure S3. Intravitreal dexamethasone injection induces ciliary epithelium proliferation. (A-B) Representative images of Pax6 IHC and EdU labeling in the ciliary body of mouse eyes exposed to (A) 0.5% DMSO vehicle, or (B) 10μM Dexamethasone. Nuclei are labeled via Hoechst staining. White arrows indicate Pax6 + EdU co-labeled cells. Dashed line indicates inset. 10 μm-thick sections. Figure S4. EdU-positive cells co-label with endothelial and microglia/macrophage markers. (A-B) Representative images of ERG IHC and EdU labeling in the ciliary body of mouse eyes exposed to (A) 0.5% DMSO vehicle, or (B) 10μM Dexamethasone. (C-D) Representative images of CD68 IHC and EdU labeling in the ciliary body of mouse eyes exposed to (C) 0.5% DMSO vehicle, or (D) 10μM Dexamethasone. Nuclei are labeled via Hoechst staining. White arrows indicate co-labeled cells. 10 μm-thick sections. Figure S5. Glucocorticoid receptor, Mineralocorticoid receptor, and 11-β-HSD1 & 2 RNA expression in RSC spheres. Transcriptomic data showing the expression of the glucocorticoid receptor (Nr3c1), mineralocorticoid receptor (Nr3c2) and the two 11-β-HSD isozymes in RSC spheres, supporting the finding of retinal precursor sensitivity to GR agonists. This graph was created from RNAseq data collected in Khalili et al. 2018. Two different mouse strains were used to generate RSC spheres that were lysed and high-quality total RNA (RIN: 9–10) was subjected to directional RNA-sequencing library construction from three independent biological replicates per mouse strain. Sequencing was performed using GAIIx (Illumina, Inc., San Diego, CA; www.illumina.com). Table S1. Compounds that met hit criteria in at least one of two screens. Table S2. Screening quality metrics. |
