Fig. 5

Nup210 Interacts with and Recruits Mef2C to NPCs

(A) Nup210 and Mef2C immunofluorescence in differentiated C2C12 cells (day 4). MHC was used as a marker for differentiated myotubes. Nuclei were stained with Hoechst. Arrowheads show quiescent myoblasts with non-detectable Nup210 or Mef2C.

(B) C2C12 cells were infected with lentiviruses carrying control and Mef2C shRNAs, induced to differentiate and stained at 72 hr after differentiation with MHC.

(C) Nup210 was immunoprecipitated from wild-type or FLAG-Mef2C expressing C2C12 myotubes, and the presence of Mef2C was determined with a pan-MEF2 antibody (top panel) or an anti-FLAG (bottom panel) antibody. Mef2C-specific band was confirmed by shRNA depletion (Figure S2B). Asterisk shows the potential Mef2A and Mef2D bands recognized by the pan-MEF2 antibody as described by the manufacturer. The specificity of these bands was not confirmed in this study. Nup210 is expressed at low levels in myotubes and not detected in the input.

(D) Differentiated C2C12 cells were analyzed by immunofluorescence against Nup210 and Mef2C (IF), and proximity ligation assays (PLA). PLA shows the interaction of Mef2C with Nup210 at the nuclear periphery (red dots). Representative images (n ≥ 3). Scale bars, 5 μm.

(E) Zebrafish embryos were co-injected with Nup210 morpholinos plus GST or Mef2Cα mRNAs and slow muscle was stained at 48 hpf.

(F) Rescue experiments from (E) were quantified by measuring somite angle (n = 10–20 embryos, performed in triplicate).

(G) Zebrafish embryos were injected with control or Nup210 morpholinos alone or in combination with Mef2Cα RNA, Pom121 MO, or Pom121 MO + Mef2Cα RNA. Slow muscle was analyzed 48 hpf.

(H) Quantification of myotome angle from (G) (n = 10–20 embryos, performed in triplicate).

(I) Control or Nup210 morpholino-depleted embryos were stained at 48 hpf for Mef2C (red dots) and F-actin (magenta). Nuclei were stained with Hoechst (blue).

(J) The localization of Mef2C in control or Nup210-depleted myotubes was analyzed by immunofluorescence. Top panel shows the downregulation of Nup210 in shRNA-depleted myotubes. Scale bar, 20 μm.

Bar plots represent mean ± SEM, n ≥ 3 replicates. ^∗p ≤ 0.05. Scale bars represent 50 μm unless otherwise specified. See also Figure S2.