Fig 1: PR3 reduces rod gene expression via Nr2e3.(A) Chemical structure of Photoregulin3 (PR3). (B) Dose-response relationship of PR1 and PR3 on Rhodopsin expression in dissociated retinal cell cultures (n = 3 for each concentration for each compound; value graphed is the mean ±SEM of 3 biological replicates). Example scans of Rhodopsin and ToPro3 staining from retinal cell cultures are shown on the right. (C) Confirmation of PR3’s potency by qPCR from intact retinal explant cultures from P4 mice treated with DMSO or 0.3 µM PR1 or PR3 for 2 days (n = 3–4 for each compound; value graphed is the mean ±SEM of the biological replicates, *p=0.0075 for DMSO vs. PR3, two-tailed t-test assuming equal variance using ?CT values). (D) Intact retinas from P11 mice were explanted in media containing DMSO or 0.3 µM PR3 for 3 DIV and then stained for S Opsin in a whole-mount preparation. Scale bar represents 50 µm. (E) PR3-treated retinas had more S Opsin+ cells per 100 µm x 100 µm field in the ventral, but not dorsal, retina compared to DMSO-treated retinas (n = 3 biological replicates; value graphed is the mean ±SEM of the biological replicates, *p=0.00063, two-tailed t-test assuming equal variance). (F) Isothermal titration calorimetric study of PR3 binding to Nr2e3. (G) Intact retinas from adult (P23–P35) wild type and Rd7 were explanted in media containing DMSO or 1 µM PR3 for 2 days and Rhodopsin expression was measured by qPCR. PR3 decreased Rhodopsin in wild type retinas but not in Rd7 retinas (n = 4 biological replicates; value graphed is the mean ±SEM of the biological replicates, *p=0.033, two-tailed t-test assuming equal variance for wild type and p=0.13, two-tailed t-test assuming equal variance for Rd7 using ?CT values).10.7554/eLife.30577.006Figure 1—source data 1.Source data for Figure 1.