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Figure 5. Reduction of NM23-X4 increases the Müller glial cell population. (A-D) shRNAs against p27Xic1 and NM23-X4 are efficient in knocking down their respective protein expression in cell culture and Xenopus embryos. The indicated short hairpin RNA (shRNA) constructs and the corresponding tagged expression constructs were co-transfected in COS7 cells and co-injected in two-cell stage Xenopus embryos. The effect was analyzed by immunoprecipitation of total lysate of cells or embryos (see Materials and methods). (E-G) Stage 41 retinal section after transfecting with pSuper vector and GFP (E), with shX4-A and GFP (F), or with shX4-B and GFP (G). (H) Enlarged view of a green fluorescent protein (GFP)-positive Müller glial cell transfected with shX4-B. (I-K) Staining against the Müller glial marker R5 at stage 41 retina transfected with shX4-B: (I) GFP; (J) R5 staining; (K) merged view. (L-N) Anti-CRALBP staining of Müller glial cells at stage 41 retina transfected with shX4-B: (L) GFP, (M) anti-CRALBP, (N) merged view. (O) Cell type distribution in the stage 41 retina transfected with the indicated construct plus GFP expressed in percentages. (P-Q) Rescue of the knock-down effect in the cell type distribution in the retina by co-introduction of shRNA construct with p27Xic1 (P) and NM23X4 (Q) expression constructs. The Müller glial cell percentages for each condition are shown. Single and double asterisks correspond to P ≤ 0.05 and 0.01, respectively; error bars indicate standard error of the mean. |
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Figure 7. Overexpression of NM23 activates gliogenesis. (A-D) Retinal section at stage 41 after lipofection with GFP (A), NM23-X1 (B), or NM23-X4 (C). A representative image of Müller glial cells in the retina transfected with NM23-X4 and GFP (D). (E-P) Immunostaining of NM23-X4 lipofected retinas with cell specific markers. (E-G) Müller glia staining using R5 antibody: (E) green fluorescent protein (GFP); (F) R5; (G) merged. (H-J) anti-CRALBP staining of Müller glial cells: (H) GFP; (I) anti-CRALBP; (J) merged. (K-M) Ganglion cell staining against islet-1 using 39.4D5 antibody: (K) GFP; (L) 39.4D5 staining; (M) merged. (N-P) Photoreceptor staining with anti-calbindin: (N) GFP; (O) calbindin staining; (P) merged. (Q) Distribution of cell types in the retina after lipofection with NM23 family members. |
