Peptide Lv enhanced cAMP production and ERK phosphorylation in chicken cone photoreceptors.

<p>(A and B) Cultured chick photoreceptors (E10+2) were treated with 500 ng/ml commercially synthesized peptide Lv or control buffer for 0, 15, 30, 60, or 120 min prior to harvest. (A) The cAMP levels increased after 15 min treatment of peptide Lv (n = 6 for each time point). *indicates that the cAMP levels at 15 and 120 min were significantly higher than 0 min (One-way ANOVA, Tukey post hoc, *<i>p</i><0.05). (B) Total ERK and pERK were detected by Western blots. The band densities were measured, and the values were calculated as a ratio of pERK to total ERK. Relative fold difference was calculated by comparison to the control, in which the control was set as 1 (n = 5 for each time point). *indicates that the pERK level at 0 min is significantly different from 30, 60, and 120 min (One-way ANOVA, Tukey post hoc, *<i>p</i><0.05). #indicates that pERK levels at both 30 and 60 min are significantly different from 15 min (One-way ANOVA, Tukey post hoc, ^#<i>p</i><0.05). (C and D) The effect of peptide Lv on pERK and cAMP is dampened by PTX. Peptide Lv increased cAMP levels (2.74±0.56) and the phosphorylation of ERK (4.37±1.09) after 4 hours of treatment (D). However, PTX blocked the effects of peptide Lv (n = 5 for each treatment). For cAMP levels, * indicates that the peptide Lv treated group is significantly higher than the control and PTX treated groups (One-way ANOVA, Tukey post hoc, *<i>p</i><0.05). For pERK levels, *indicates that the peptide Lv treated group is significantly higher than all other groups, while #indicates that the peptide Lv+PTX group is significantly higher than the control and PTX treated groups (One-way ANOVA, Tukey post hoc, both *and #indicate <i>p</i><0.05).</p

Synthesized peptide Lv increased the mRNA and protein expression of L-VGCCα1 subunit in cone photoreceptors.

<p>Cultured E18 (E16+2) photoreceptors were treated with 500 ng/ml synthetized peptide Lv for 0 (control, filled square), 15 (gray circle), or 30 min (triangle). Averaged current-voltage (I–V) relationships were obtained from the step command as in current density (pA/pF) and membrane voltage (mV). (B) Treatment with peptide Lv for at least 3 hrs elicited an increase of L-VGCCα1 subunit expression. * indicates that treatment with peptide Lv for 3 or 4 hrs is significantly different from the other groups (One-way ANOVA, Tukey post hoc, *<i>p</i><0.05). (C) Treatment with peptide Lv for 4 hrs (dark gray) significantly increased the mRNA levels of both α1C and α1D compared with the control (light gray, n = 6 for each group). * indicates the statistical difference between the peptide Lv treated group and control (Student’s <i>t</i>-test; *<i>p</i><0.05). (D) Compared to the treatment of peptide Lv alone (filled circle), treatment with 0.5 µg/ml PTX did not dampen the effect of peptide Lv (triangle). (E and F) The effect of peptide Lv was developmental age dependent. Chicken cone photoreceptors at E12 (E10+2) or E18 (16+2) were cultured and treated with peptide Lv. Peptide Lv significantly increased current density at E12 (E12: control, −4.49±0.66 pA/pF, n = 9, open square; peptide Lv treated cells, −7.22±0.45 pA/pF, n = 16, dark square. E18: control, −8.40±1.11 pA/pF, n = 9, open triangle; peptide Lv, −10.93±0.70 pA/pF, n = 17, dark triangle). Comparisons between the control and peptide Lv treated groups were made using Student’s <i>t</i>-test; *<i>p</i><0.05.</p

Sequence alignment of propeptide Lv among different species.

<p>Mouse, rat, human, and chicken peptide Lv proprotein were over 80% homologous. The sequence contained a signal peptide sequence (green), proprotein convertase cutting motifs (red), the main peptide Lv coding region (blue), and a transmembrane domain (purple). Peptide II (control for the electrophysiological studies) is highlighted in orange. Highly conserved identical sequences are denoted by (<b>*</b>), conserved substitution by (<b>:</b>), and semiconserved substitution by (<b>.</b>).</p

A flow chart illustration of the <i>in silico</i> computational screening strategy and procedure.

<p>Human and mouse full length protein databases were subjected to five step separation computer algorithms including N-terminal signal peptide sequence (secretion marker), propeptide convertase targeting motif, transmembrane domain, potential glycosylation modification, and sequence homology across species. Potential candidates were ranked according to the score assigned from each step (Data S1).</p

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