Figure 6.

Figure 6.

(A) An experimental timeline that describes the injury, MO injection, BrdU pulse, late electroporation of the retina, and EdU pulse 3 h before harvest at 8 dpi. (B, C) IF confocal microscopy images of retinal cross sections show increased BrdU⁺ MGPCs at 8 dpi in oct4 knockdown from fifth day onwards and a proof of the delay in quitting cell cycle revealed by EdU co-labeling with BrdU⁺ MGPCs (B), which is quantified (C); *P < 0.001, N = 4. White arrowheads mark BrdU⁺/EdU⁺ cells in (B). (D) qRT-PCR analysis of Tgf-β signaling component genes and its reporter tgfbi mRNA levels in late oct4 knockdown retina, at 8 dpi; *P < 0.02 (t test), N = 4. (E) qRT-PCR analysis of snail family genes’ mRNAs in late oct4 knockdown retina, at 8 dpi; *P < 0.03 (t test), N = 4. (F) qRT-PCR analysis of ascl1a, mycb, oct4, sox2, lin28a, mmp2, and mmp9 mRNA levels in late oct4 knockdown retina, at 8 dpi. (G) qRT-PCR analysis of let-7a miRNA levels show a decline because of oct4 late knockdown in 8 dpi retina. (H, I) Western blot analysis of various regeneration-associated factors in late oct4 knockdown retina at 8 dpi, which is quantified by densitometry (I). Gapdh is used as the loading control. Ctl MO is control MO. Error bars are SD. (B) Scale bars, 10 μm; the asterisk marks the injury site; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer (B).