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The increased MGPCs seen in late <italic>oct4</italic> knockdown in regenerating retina is caused by the delay in cell cycle exit.(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).
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