Fig 1: VEGF-related miRNAs and VEGF levels in photoreceptors. (A) qRT-PCR for cellular miRNAs miR-20a-3p, miR-20a-5p, miR-20b, miR-106a-5p expression determination and (B) ELISA test for VEGF levels in photoreceptors stimulated with normal glucose (5 mM D-glucose); high glucose (30 mM D-glucose); HG + RvD1 (RvD1, 50 nM); HG + RvD1 + Boc2 (20 μM). Values are expressed as mean ± s.e.m. of n = 9 values, obtained from the triplicates of three independent experiments. They were analyzed by one-way ANOVA followed by Bonferroni’s test for each panel. NG, normal glucose; HG, high glucose; RvD1, Resolvin D1; Boc2, selective FPR2 inhibitor. *P < 0.01 vs. NG; °P < 0.01 vs. HG.
Fig 2: Nanosight and TEM of exosomes released from photoreceptors. Size-distribution of exosomes assessed using a Nanoparticle Tracking Analysis (A) and their release into the extracellular medium assessed by electron microscopy (B) in photoreceptors stimulated with normal glucose (5 mM D-glucose); high glucose (30 mM D-glucose); HG + RvD1 (RvD1, 50 nM); HG + RvD1 + Boc2 (20 µM). Scale bar 500 nm. Magnification 2000X. Values are expressed as mean ± s.e.m. of n = 9 values, obtained from the triplicates of three independent experiments. They were analyzed by one-way ANOVA followed by Bonferroni’s test for each panel. NG, normal glucose; HG, high glucose; RvD1, Resolvin D1; Boc-2, selective FPR2 inhibitor. *P < 0.01 vs. NG; °P < 0.01 vs. HG.
Fig 3: FPR2 and RvD1 levels, cell viability, ROS content, NF-kb protein expression. (A) ELISA detecting the levels of FPR2 receptor and (B) constitutive Resolvin D1 before and after RvD1 addition to photoreceptors exposed to high glucose; (C) XTT assay for determination of total cell number; (D,E) average intensity from DCFH-DA for total intracellular ROS levels compared to a negative control (yellow) and a positive control (fill red, 100 µM H2O2). Green = normal glucose; black = high glucose; blue = HG + RvD1 and red = HG + RvD1 + Boc2; (F) Western Blotting determination and representative images of NF?B protein levels into photoreceptors stimulated with normal glucose (5 mM D-glucose); high glucose (30 mM D-glucose); HG + RvD1 (RvD1, 50 nM); HG + RvD1 + Boc2 (20 µM). Values are expressed as mean ± s.e.m. of n = 9 values, obtained from the triplicates of three independent experiments. They were analyzed by one-way ANOVA followed by Bonferroni’s test for each panel, except for panel (B) were ANOVA for repeated measures was applied. NG, normal glucose; HG, high glucose; RvD1, Resolvin D1; Boc-2, selective FPR2 inhibitor. *P > 0.01 vs. NG; °P > 0.01 vs. HG.
Fig 4: Exosomal miRNAs and VEGF levels. (A)qRT-PCR for miR-20a-5p, miR-20a-3p, miR-20b, miR-106a-5p expression and (B) ELISA test for VEGF levels from exosomes released after stimulation of photoreceptors with normal glucose (5 mM D-glucose); high glucose (30 mM D-glucose); HG + RvD1 (RvD1, 50 nM); HG + RvD1 + Boc2 (20 µM). Values are expressed as mean ± s.e.m. of n = 9 values, obtained from the triplicates of three independent experiments. They were analyzed by one-way ANOVA followed by Bonferroni’s test for each panel. NG, normal glucose; HG, high glucose; RvD1, Resolvin D1; Boc-2, selective FPR2 inhibitor. *P < 0.01 vs. NG; ° P < 0.01 vs. HG.
Fig 5: Annexin A2 (A) and Flottilin-1 (B) labeling of exosomes released from primary photoreceptors stimulated with normal glucose (5 mM D-glucose); high glucose (30 mM D-glucose); HG + RvD1 (RvD1, 50 nM); HG + RvD1 + Boc2 (20 μM). Values are expressed as mean ± s.e.m. of n = 9 values, obtained from the triplicates of three independent experiments. They were analyzed by one-way ANOVA followed by Bonferroni’s test for each panel. NG, normal glucose; HG, high glucose; RvD1, Resolvin D1; Boc-2, selective FPR2 inhibitor. *P < 0.01 vs. NG; °P < 0.01 vs. HG.
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