Extended Data Fig. 6: Protective and regenerative effect of OSK is dependent on TET1 and TET2.
From: Reprogramming to recover youthful epigenetic information and restore vision
a, Mouse Tet1, Tet2 and Tet3 mRNA levels with or without OSK expression in RGCs (n = 6 biological replicates each condition). The P value indicated with an asterisk was calculated using an unpaired one-tailed t-test. b, Representative images of retinal wholemounts transduced with AAV2-tTA;TRE-OSK in combination with a AAV2-shRNA-YFP (yellow fluorescent protein) having either a scrambled sequence (sh-Scr) or a hairpin sequence to knockdown Tet1 (sh-Tet1) or Tet2 (sh-Tet2) expression, at titre ratio 5:5:1. Retinal wholemounts immunostained for Klf4. n = 3 retinas for each condition. c, Quantification of shRNA-YFP AAV transduction in OSK-expressing RGCs (n = 3 retinas for each condition). d, Mouse Tet1, Tet2 and Tet3 mRNA levels with sh-Scr (n = 5), sh-Tet1 (n = 4) or sh-Tet2 (n = 5) YFP AAV2 in RGCs in the presence of OSK expression. e, f, Quantification of axon regeneration (e, n = 4 eyes each condition) and RGC survival (f, n = 10, 7 and 9 eyes) at 2 wpc in retinas co-transduced with AAV2- tTA;TRE-OSK;shRNA. g, Mouse Stat3 mRNA levels after knockdown using sh-Scr (n = 5), sh-Tet1 (n = 4) or sh-Tet2 (n = 5) in RGCs in the presence of OSK expression. h, Cre-dependent Tomato expression in RGCs after intravitreal AAV2-Cre injection of Tomato reporter mice (Rosa-CAG-lox-STOP-lox-Tomato), and the co-expressed frequency of Cre and Klf4 (n = 3 eyes). i, j, RGC survival (i) and representative longitudinal sections of regenerating axons in longitudinal sections (j) in response to OSK expression (AAV2-tTA;TRE-OSK, n = 5 for each condition) compared to no expression (saline, n = 3 and 4), 16 days after crush injury in Tet2flox/flox mice injected with saline (Tet2 WT) or AAV2-Cre (Tet2 cKO). Scale bars (b, h and j), 100 μm. Two-way ANOVA in a, d, i; unpaired two-tailed Student’s t-test in g; one-way ANOVA in e, f. All data are mean ± s.e.m.
