Article Figures & Data
Figures
- Figure 1.
Expression and purification of rhMANF. The 6xhis-tagged protein was expressed in E. coli and purified on nickel columns. Purified rhMANF was detected as a single band ∼20 kDa when electrophoresed on 4–12% NuPAEG gel and visualized with Coomassie Blue.
- Figure 2.
Expression of MANF in the retina. A, Expression of MANF during postnatal development. Retinas were collected at designated time points from wild-type Sprague Dawley rats. Levels of MANF were detected by Western blot analysis with anti-MANF antibodies. High levels of MANF expression were detected during postnatal development (from PD1 to PD16; A, upper panel). As the retinas mature, the expression decreases (from PD25 to PD60; A, upper panel). The same blot was reprobed with anti-β-actin antibodies as loading controls (A, lower panel). Cryosections of 12 µm of eyes of PD20 wild-type Sprague Dawley rats along the vertical meridian were stained with anti-MANF antibodies (B, D, E). Müller cells were identified by antibodies against GS (C–E). Extensive colocalization of MANF and GS immunoreactivities (D, E, yellow) indicates that MANF in the retina is expressed in Müller cells. RPE cells, neurons in the GCL, and neurons in the INL also display MANF immunoreactivities (B, D, white arrowheads in E). OS, outer segments; IS, inner segments; OPL, outer plexiform layer; IPL, inner plexiform layer. Scale bar: 50 µm (D) and 20 µm (E).
- Figure 3.
Protection of rod photoreceptors by MANF. The right eyes of S334ter-3 rats were treated with 6 µg (in 3 µl of PBS) of MANF by intravitreal injection, and the left eyes were injected with of PBS. Tissues were collected at PD20. Semi-thin sections were cut and stained with toluidine blue. The ONL in the control eye had only one row of nuclei in the superior retina (between arrowheads, A), whereas in the eyes treated with MANF, there are three to four rows of nuclei in the ONL (between arrowheads, B). In comparison, the ONL in a normal wild-type rat at PD20 contains 13–14 rows of nuclei (C). Quantitative analysis shows that the ONL in the MANF-treated retinas (superior retina) is significantly thicker than that in the PBS-treated retinas (D, triple asterisks indicate p < 0.001, Student’s t test). OS, outer segments; IS, inner segments; OPL, outer plexiform layer. Scale bar: 25 µm.
- Figure 4.
Protection of cone photoreceptors by MANF. The right eyes of S334ter-3 rats were treated with 6 µg (in 3 µl of PBS) of MANF by intravitreal injection, and the left eyes were injected with of PBS at PD20. Eyes were harvested 10 d after injection (PD30). Retinas were stained with PNA to identify cone outer segments. In the control retinas treated with PBS, many PNA-negative patches are present (A). In MANF-treated retinas, PNA-negative patches were very small and in many areas are not present (B). Quantitative analysis of PNA-positive cells in the superior retinas are significantly more in MANF-treated eyes (569.5 ± 46.5, n = 6) than in PBS-treated eyes (398.7 ± 25.4, n = 6; C, triple asterisks indicate p < 0.001, Student’s t test). Scale bar: 100 µm.
- Figure 5.
Preservation of ERG by MANF. The right eyes of rd 10 mice were treated with 2 µg (in 1 µl of PBS) of MANF by intravitreal injection at PD18. The left eyes were treated with PBS. ERGs were recorded from both eyes simultaneously 10 d later at PD28. Scotopic b-waves evoked by white light flashes of −0.4 log cd-s/m2 were reliably recorded but the a-waves were undetectable (A). Significantly higher b-wave amplitudes were recorded from MANF-treated eyes, with an average amplitude of 115 ± 16.5 μV (n = 5), as compared with PBS-treated eyes whose average amplitude is 82.4 ± 10.7 μV (n = 5; B, double asterisks indicate p = 0.0017, Student’s t test).
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