Fig 1: Norgestrel upregulates fractalkine-CX3CR1 signaling in the rd10 retina.(A) 661W cells were serum-starved and treated with 20 μM Norgestrel (Norgestrel) or the equivalent DMSO control (Control) over 6 and 24h. RT-qPCR analysis detected a significant increase in mRNA levels of fractalkine in serum-starved cells treated with Norgestrel. N = 6 biological replicates, n = 3 technical replicates per sample. (B) Norgestrel-supplementation in rd10 mice induced a significant increase in fractalkine and CX3CR1 mRNA expression in P20-P30 retinas compared to control. Control column represents age-matched rd10 mice on control diet. N = 3 mice per group, n = 3 technical replicates per mouse. (C) Western blot for fractalkine at P20 and P25 confirmed an increase in full-length fractalkine (100kDa; red box) at both time-points with Norgestrel treatment. Total protein stain shows similar loading between samples. Blots are representative of 6 biological replicates per group. (D) Fluorescent microscopic images of fractalkine (green) in the central retina from P15-40 of control and Norgestrel-fed rd10 mice. Fractalkine staining is evident in the outer nuclear layer (ONL) of the retina in Norgestrel-supplemented mice. Hoechst (blue) staining reveals the cell layers in the retina. Scale bar 50μm. N = 3 mice per group, n = 3 sections per mouse. (E) 661W cells were cultured in the absence of serum (serum-starved 661W) over 3 h. 661W cells were then placed in culture with microglia that had been treated with (Microglia Fract.) or without (Microglia) 1.2μg/ml recombinant full-length fractalkine over 24h. Co-cultures were incubated for a further 24h. Apoptosis of 661W cells was detected by TUNEL of DNA strand breaks in cell nuclei. Co-cultures with fractalkine-treated microglia had less TUNEL-positive 661W cells than equivalent vehicle control. Scale bar 30μm. (F) Quantification of TUNEL-positive 661W cells cultured in the absence of serum and following co-incubation with primary rd10 microglial cells pre-treated with recombinant full-length fractalkine or vehicle. N = 9 mice for primary culture, n = 5 technical replicates per group. Results are presented as mean ± SEM (t-test, *p<0.05, ***p<0.005, ****p<0.001).