Fig 2: Post-injury Marcks overexpression also promotes RGC axon regeneration.A Top: experimental timeline. Bottom: representative image of optic nerve showing that post-injury overexpression of Marcks in RGCs induced significant optic nerve regeneration 2 weeks after optic nerve crush. The right columns showed enlarged images of nerves at 500, and 1000 μm distal to the crush sites marked by white boxes on the left. Scale bar: 200 μm. *: crush sites. B Quantification of axon regeneration in (A). **p < 0.01, *p < 0.05, ns no significance. n = 5 mice in each group. C Representative images of retinal sections from intact retina or injured retina 2 weeks after injury stained with RBPMS. Scale bar: 20 μm. D Quantification of RGC survival in (C) showing that Marcks overexpression did not affect RGC survival. ns no significance. RGCs were analyzed from at least 7 non-adjacent retinal sections for each animal, from 4 mice per group.

Fig 3: Marcks overexpression in RGCs induces significant and persistent optic nerve regeneration.A Top: experimental timeline. Bottom: representative image of optic nerve showing that overexpression of Marcks in RGCs induced drastic optic nerve regeneration 2 and 4 weeks after optic nerve crush. The right columns showed enlarged images of nerves at 500, 1000, 1500, and 2000 μm distal to the crush sites marked by white boxes on the left. Scale bar: 200 μm. *: crush sites. B Quantification of axon regeneration in (A). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns no significance. n = 6–9 mice in each group. C Representative images of retinal sections from intact retina or injured retina 2 and 4 weeks after optic nerve crush stained with RBPMS. Scale bar: 20 μm. D Quantification of RGC survival in (C) showing that Marcks overexpression did not affect RGC survival, while fewer RGC survived for 4 weeks after optic nerve crush compared to 2 weeks. ****p < 0.0001, ns no significance. RGCs were analyzed from at least 7 non-adjacent retinal sections for each animal, from 4 mice per group.

Fig 4: Marcks levels are downregulated during development and after optic nerve crush.A Representative images of retinal sections from C57BL/6 mice of different ages (0, 7, 21, and 56 days postnatal) showing gradually reduced levels of Marcks in RGCs during development. Retinal sections were stained with RBPMS (red), and MARCKS (green). Scale bar: 20 μm. B Quantification of fluorescence intensity of MARCKS in RGCs shown in (A). ****p < 0.0001, *p < 0.05. RGCs were analyzed from at least 7 non-adjacent retinal sections for each animal, from 6 mice per group. C Representative images of retinal sections showing decreased Marcks levels in RGCs after optic nerve crush. Retinal sections were stained with RBPMS (red), and MARCKS (green). Scale bar: 20 μm. D Quantification of fluorescence intensity of MARCKS in RGCs shown in (C). ns no significance, *p < 0.05. RGCs were analyzed from at least 7 non-adjacent retinal sections for each animal, from 6 mice per group.

Fig 5: Marcks overexpression does not induce changes in transcripts with axon regeneration.A Principal component analysis of samples demonstrating the similarity between Marcks overexpression and control groups 3 days after optic nerve injury. B Hierarchical clustering of samples showing the similarity between Marcks overexpression and control groups. The value in each grid represents the Euclidean distance between two samples. C Pairwise correlations of samples showing that Marcks overexpression had little impact on gene transcription of RGCs. The upper right showed Pearson correlation coefficient between pairwise samples. The lower left gave their scatter plots of normalized counts. D GO analysis of differentially expressed genes, the terms with the 10 highest log10 (P-value) were shown as a bar plot. Results showing that Marcks overexpression did not induce changes in transcripts with roles in axon regeneration.