Figure 5: An intact P1-1 element is required for HuR-mediated regulation of NPM expression.

(a) Schematic diagram of the Renilla luciferase reporters used in the experiments described in a–f. (b,c) Exponentially growing C2C12 cells were transfected with plasmids expressing Rluc, Rluc-NPM-3′ or Rluc-NPM-3′-mut1. Total extracts from these cells were prepared 24 h after transfection and used for IP using the anti-HuR antibody (3A2) or IgG as a control. (b) Western blot analysis with the anti-HuR antibody was performed using these IP samples. (c) Rluc mRNA associated with the immunoprecipiated HuR was determined by RT-qPCR. The relative Rluc RNA levels from Rluc-NPM-3′ and Rluc-NPM-3′-mut1 were plotted relatively to the Rluc RNA reporter±s.e.m. of three independent experiments. (d,e) The expression levels of these Rluc RNA reporters were also determined by RT-qPCR in C2C12 cells depleted or not of HuR (d) or overexpressing GFP or GFP-HuR (e). The expression levels of the Rluc mRNAs were normalized over total Rluc DNA transfected as described⁵⁹ and then standardized against GAPDH mRNA level±s.e.m. of three independent experiments **P<0.001; NS, non significant (t-test). (f) C2C12 cells were transfected with the Rluc reporter RNA described above in the presence of AP-GST or AP-HuR-GST proteins. The stability of these reporter Rluc mRNAs was determined by AcD pulse-chase experiments. The percentages shown were plotted±s.e.m of three independent experiments. (g) Schematic diagram of GFP or the GFP-conjugated full-length NPM isoforms: WT NPM (GFP-NPM-3′) and the NPM 3′-UTR with the first AU-rich sequence mutated (GFP-NPM-3′-mut1). The coding region of NPM is shown as NPM CDR. (h–j) C2C12 cells expressing these isoforms or the GFP-HuR protein were fixed on day 3 of muscle cell differentiation. (h) Phase contrast images of the myotubes described above. (i) Immunofluorescence (using the anti-My-HC antibody) images of these myotubes. (j) Fusion index±s.e.m of three independent experiments, **P<0.001, ***P<0.0001 (t-test). Images of a single representative field are shown. Scale bar, 50 μm (h) and 10 μm (i).