Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 2

Figure 2. miR-211 controls Ezrin expression in vivo

(A) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm.

(B) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated on the X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. Mann and Whitney test *p ≤ 0.05 (n=3 mice for each time point). Values from 12h dark-adapted mice (T0) were set to one.

(C) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old miR-211-/- and WT control mice sacrificed 3h after light on at 10 AM (diurnal condition). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm.

(D) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).

(E) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR-211-/- mice. The graph shows the quantification of Ezrin normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=5 mice) Mann and Whitney test (miR-211-/- vs WT), **p ≤ 0.01.

(F) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old miR-211-/- mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one.

(G) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one.

(H) qRT-PCR assay for Mtmr10 and Klf13 from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after on at 10 AM. The graph shows the expression level of these genes normalized to the Hprt. Bar graphs represent mean values ± s.e.m. (n=3 mice)

Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.