Fig. S2

Trip6, Mef2C and Pom121 depletion in Zebrafish embryos. Related to Figures 4 and 5.

(A) Wild-type or Trip6 CRISPR-generated mutants were stained with the F59 antibody at 48 hpf. Slow muscle was imaged by confocal microscopy. Representative image of a maximum projection of 30-40 sections. n ≥ 10 embryos. (B) C2C12 cells were infected at 36 hours post-differentiation with lentivirus carrying control or MEF2C-specific shRNAs. Cells were collected 72 hours post-infection and Mef2C depletion was analyzed by western blot using an anti-Mef2 antibody. According to the manufacturer anti-Mef2 recognizes Mef2A, C and D. Western blots show the specific Mef2C protein band. The specificity for Mef2A and D was not confirmed (C) The interaction of the nucleoproin Nup210 (green) with nucleoporins recognized by the mAb414 antibody, were analyzed by proximity ligation assays. PLA interactions are shown in red. (C) MEF2Cα was depleted during Zebrafish development using specific morpholinos previously characterized (Hinits and Hughes, 2007). Slow muscle was stained at 48 hpf and compared to Control or Nup210-depleted animals. Representative image of a maximum projection of 30-40 sections. n = 10-20 embryos by triplicate. (E) The transmembrane Pom121 nucleoporin was depleted during Zebrafish development and animals were analyzed at 48 hpf. No muscle alterations are observed in the absence of Pom121. Representative image of a maximum projection of 30-40 sections. n = 10-20 embryos by triplicate. (F) Due to the absence of antibodies that recognize Zebrafish Pom121 we verified the effectiveness of Pom121 morpholinos by determining their ability to down-regulate the expression of a fusion RNA containing Pom121 5’UTR upstream of the RFP coding sequence as described by Veldman et al. Pom121 morpholinos efficiently block the translation of the fluorescent reporter (n = 6-10 embryos by duplicate).