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. 2019 Feb 4;218(2):489–507. doi: 10.1083/jcb.201802113

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© 2019 Mitra et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 5. — Insm1a inhibits mycb expression in regenerating retina. (A) MO-based gene knockdown of ascl1a down-regulates mycb:gfp-luciferase expression in embryos. *, P < 0.002. (B) Ascl1a overexpression inhibits mycb:gfp-luciferase expression in embryos. *, P < 0.0002. (C) MO-mediated ascl1a inhibition in retina as early as 8 h after injury causes an increase in mycb, but not myca expression. *, P < 0.009; n = 3 biological replicates. n.s., not significant. (D) Diagram of mycb promoter with putative Insm1a binding site. (E) MO-based insm1a knockdown significantly up-regulated both myca and mycb expression in injured retina at 2 dpi. *, P < 0.001. (F) The insm1a knockdown through MO up-regulates mycb:gfp-luciferase expression in zebrafish embryos by luciferase assay. *, P < 0.0001. (G) Insm1a overexpression inhibits mycb:gfp-luciferase expression in embryos. *, P < 0.001. (H) Schematic of mycb promoter with mutated Insm1a-binding site. (I) Insm1a inhibition through MO has no effect on mutated mycb:gfp-luciferase expression in zebrafish embryos by luciferase assay. n.s., not significant. (J and K) Myc inhibition through MO (J) and 10058-F4 (K) cause significant down-regulation of insm1a expression in 2-dpi retina. (L) 10058-F4–based Myc blockade inhibits insm1a:gfp-luciferase in embryos. Promoter activity is normalized light units with internal control Renilla luciferase. *, P < 0.0002. Error bars are SD. BS, binding site.