Extended Data Fig. 5: OSK activates Stat3 in the absence of mTOR activation or global demethylation.
From: Reprogramming to recover youthful epigenetic information and restore vision
a, Representative images of retinal wholemounts transduced with AAV2-tTA (−OSK) or AAV2-tTA;TRE-OSK (+OSK) in the presence or absence of crush injury after 3 days. Retinal wholemounts immunostained for pStat3, Klf4 and RBPMS. n = 2 retinas each condition. b, Representative images of retinal wholemounts transduced with d2EGFP- or OSK-encoding AAV2 in the presence or absence of a crush injury. Retinal wholemounts immunostained for RBPMS and mTOR activation marker phosphorylated S6 (pS6). n = 4 retinas for each condition. c, Percentage of pS6-positive RGCs in intact and crushed samples (n = 4 retinas for each condition). d, Representative images of d2EGFP in retina expressed from the Tet-On AAV system. No GFP expression was observed in the absence of DOX. GFP expression reached peak levels 2 days after DOX induction and remained at a similar level at day 5 after induction. n = 2 retinas each condition. e, Representative images of retinal d2EGFP expression using the Tet-Off AAV system with various durations of DOX treatments (2 mg ml−1). Once pre-treated with DOX to suppress expression (on DOX), GFP was sparse even on day 8 after DOX withdrawal, lower than peak expression (Never DOX). n = 2 retinas each condition. f, Axon regeneration at 2 or 4 wpc in response to OSK induction either pre- or post-injury (n = 4, 5, 5, 4 and 4 eyes, respectively). g, Correlation between ribosomal DNA methylation (DNAm) age and chronological age of sorted mouse RGCs (1 month, n = 6; 12 months, n = 2; 30 months, n = 5), with the light blue region representing the confidence interval. P value of the linear regression is calculated by two-sided F-test of overall significance. In agreement with previous studies, DNA methylation age estimates of neurons tend to be lower than their chronological age but remain correlated (see Methods). h, Average DNA methylation levels across the mouse genome in RGCs from different ages and treatments, based on 703,583 shared CpG sites from RRBS of all samples (combined strands), n = 6, 8, 2, 8, 6, 4, 8, 8, 6, 5, 6, 4 and 5, respectively. i, Correlation of DNA methylation at each CpG site versus age (x-axis; 1 month, 12 months, 30 months) and versus injury (y-axis; intact, injured GFP). The heat map represents the number of sites located in each block of value coordinates. Pearson’s correlation coefficient, r = 0.34, P < 1e−200. j, Hierarchical clustered heat map of methylation levels of 4,106 CpGs that significantly changed in RGCs after crush injury (intact vs injured GFP, q < 0.05) and the effect of OSK. k, Top biological processes associated with the 698 CpGs that were significantly altered by both injury and OSK. Two-way ANOVA with Bonferroni correction in c, f. Scale bars (a, b, d, e), 100 μm. All data are mean ± s.e.m.
