Supplementary Material

<p>Supplemental material, Supp_Figure_(1) for Circadian Regulation of Mitochondrial Dynamics in Retinal Photoreceptors by Janet Ya-An Chang, Liheng Shi, Michael L. Ko, and Gladys Y.-P. Ko in Journal of Biological Rhythms</p

Overexpression of miR-150 decreased VEGFR2 protein level in endothelial cells.

<p>The HUVE cells were transfected with miR-150 (has-miR-150) or a scramble microRNA (Scramble) and cultured for an additional 60 hr. The protein levels of c-Myb and VEGFR2 are significantly lower in HUVE cells with overexpression of miR-150 compared to the scramble (student’s <i>t</i>-test; *<i>p</i> < 0.05). n = 4 for each group. (B) The protein level of VEGFR2 is significantly lower in HRECs transfected with has-miR-150 compared to the ones transfected with scramble (student’s t-test; *p < 0.05). n = 4 for each group. (C) The retinas from WT and miR-150^-/- mice under normal chow diet (Normal) or HFD were isolated and processed for Western blotting. Some retinas were trypsin-digested to obtain the retinal vasculature followed by immunostaining with the VEGFR2 antibody conjugated with Alexa-488. The scale bar = 100 μm.</p

Dark-adapted (scotopic) retinal light responses (Data for Fig 2A, 2B and 2E).

<p>Dark-adapted (scotopic) retinal light responses (Data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.g002" target="_blank">Fig 2A, 2B and 2E</a>).</p

Deletion of miR-150 exacerbates HFD-induced DR neovascularization and microaneurysms.

<p>(A) Upper two rows: the whole mount retinal vasculature was stained with FITC-labeled isolectin-B4. The first row: the fluorescent images from 4 experimental groups were taken at 5X (scale bar = 400 μm). The highlighted regions (yellow square) were magnified at 10X and displayed in the second row. The second row: the fluorescent images from 4 experimental groups were taken at 10X (scale bar = 100 μm). Lower two rows: the mouse retinas were trypsin-digested and the retinal vasculatures were stained with hematoxylin and eosin. The third row: the whole retinal vasculature images are shown (scale bar = 400 μm). The fourth row: magnified images of retinal vasculature (from the third row) are shown (scale bar = 100 μm). White arrowheads indicate the pericytes, the red arrowheads indicate the acellular capillaries, and the white circles indicate the microaneurysm-like (vascular extrusion) structures. (B) Wild type and miR-150^-/- mice fed with HFD have significantly higher (*) vasculature in central and peripheral retinal areas compared to mice fed with normal chow diet (Normal). There is a statistical significant difference in the interaction between miR-150 null mutation and HFD regimen (2-way ANOVA; #). (C) Wild type and miR-150^-/- mice fed with HFD have significantly higher (*) densities of microaneurysm-like structures (microaneurysms per 0.6 mm² retinal area) compared to mice fed with normal chow diet (Normal). There is a statistical significant difference in the interaction between miR-150 null mutation and HFD regimen (2-way ANOVA; #). WT-Normal: n = 6; WT-HFD: n = 8; miR-150^-/--Normal: n = 6; miR-150^-/--HFD: n = 8. <i>p</i> < 0.05 (denoted as *, #).</p

Scotopic and photopic light responses are decreased in WT and miR-150^-/- mice fed with HFD.

<p>All mice were dark adapted for at least 6 hours before ERG recordings. (A) The average scotopic ERG a-wave amplitudes recorded from miR-150^-/- with HFD (miR-150^-/--HFD) are significantly lower compared to the WT fed with normal chow diet (WT-Normal; *) or miR-150^-/- fed with normal chow diet (miR-150^-/--Normal; #). There is no statistical difference between WT fed with HFD (WT-HFD) and miR-150^-/--HFD. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) a-wave amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). The miR-150^-/- mice (both normal chow and HFD groups) had significantly smaller (<b>w</b>) a-wave amplitudes compared to the WT mice (both normal chow and HFD groups). (B) The averaged photopic ERG a-wave amplitudes recorded from WT-HFD are significantly lower than WT-Normal (*) at 3 and 10 cd.s/m² light intensities. The photopic a-wave amplitudes recorded from miR-150^-/--HFD are significantly lower than WT-normal (#) at 3, 10, and 25 cd.s/m² light intensities. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). (C) The average scotopic ERG b-wave amplitudes recorded from miR-150^-/--HFD are significantly lower compared to WT-Normal (*) or miR-150^-/--Normal (#). There is no statistical difference between WT-HFD and miR-150^-/--HFD. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) a-wave amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). The miR-150-/- mice (both normal chow and HFD groups) had significantly smaller (<b>w</b>) a-wave amplitudes compared to the WT mice (both normal chow and HFD groups). (D) The averaged ERG photopic b-wave amplitudes recorded from WT-HFD are significantly lower than WT-Normal (*) and miR-150^-/--Normal (#) at 1 and 10 cd.s/m² light intensities. The photopic b-wave amplitudes recorded from miR-150^-/--HFD are significantly different from the other 3 groups (&) at 25 cd.s/m² light intensities. HFD-mice (both WT and miR-150^-/- groups) had significantly smaller (<b>v</b>) amplitudes compared to mice fed with a normal chow (both WT and miR-150^-/- groups). (E) The averaged scotopic oscillatory potential amplitudes [as a summation from OP1 to OP4; Ʃ(OP1-4)] recorded from HFD-mice (both WT-HFD and miR-150^-/--HFD) are significantly lower (*) than mice fed with a normal chow (both WT-Normal and miR-150^-/--Normal) at 0.1, 0.3, 10, and 25 cd.s/m² light intensities. (F) The averaged photopic oscillatory potential amplitudes [Ʃ(OP1-4)] recorded from HFD-mice (both WT-HFD and miR-150^-/--HFD) are significantly lower (*) than mice fed with a normal chow (both WT-Normal and miR-150^-/--Normal) at 25 cd.s/m² light intensities. (A-F) Overall, there is no statistical significance of interaction between two factors: miR-150 null mutation and HFD regimen (2-way ANOVA). <i>p</i> < 0.05 (denoted as *, #, &, v, w). Data of scotopic and photopic ERG a- and b-waves and OPs are listed in Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.t001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157543#pone.0157543.t002" target="_blank">2</a>.</p