Fig. S3

mmp9 expression pattern and impact of SHH protein injection in regenerating retina. (A-C) BF and IF microscopy images of mmp9 and BrdU⁺ cells at various time points post injury (A), and BrdU co-labeling with mmp9 at 5dpi (B), quantified in (C). (D,E) ISH microscopy revealed increased mmp9 expression in 4dpi retina with cyclopamine treatment (D), mRNA levels quantified in (E). (F) Quantification of mmp9⁺ cells in 5% (v/v) DMSO control, 10μM cyclopamine treatment and shha or sufu knockdowns in 4dpi retina. (G) IF microscopy images show Salvianolic acid B and SB-3CT dependent decline in GFP⁺ MGPCs in 1016 tuba:GFP transgenic zebrafish at 4dpi. (H) MOs against control and mmp9 were injected and electroporated at 2dpi, then an i.p. injection of BrdU was given on 4dpi, 3 hours before euthanasia, and no change in the number of BrdU⁺ cells was found in both knockdowns. (I) Quantification of ascl1a⁺ and mmp9⁺ cells in control and ascl1a knockdown retina at 4dpi. (J) Western blotting assay indicating cyclopamine or mmp9 knockdown in 2.5dpi retina caused decline in Shha expression levels. (K) Zone of BrdU⁺ cells in the regenerating retina, increased upon injection of recombinant Shha protein (200ng) at 4dpi. 0(L,M) IF microscopy images revealed an increase in BrdU⁺ cell number in combined injection of recombinant SHH protein and shha MO, and isolated injection of SHH protein (200ng) in 4dpi retina, whereas BrdU⁺ cells declined in shha knockdown (L), which is quantified in (M), suggesting external SHH could impact retina regeneration even in absence of endogenous Shh protein. (N) RT-PCR analysis of Ascl1 and Lin28a genes in 6dpi mouse retina exposed to recombinant SHH protein at the time of injury until harvest. Scale bars, 10 μm (A,B,G,H,K,L). Error bars are SD.