Figure 3: HuR regulates the stability of the NPM mRNA.

(a) Total extracts from C2C12 myoblasts treated with siRNA HuR or siRNA Ctr were used for western blot analysis to detect NPM, HuR and α-tubulin (loading control). Representative gel of three independent experiments. (b) RNA was prepared from C2C12 cells transfected with HuR or Ctr siRNAs (for 48 h), and then treated with ActD for 0, 3, 6 or 9 h. Northern blot analysis was performed using radiolabelled probes against NPM mRNA and 18S rRNA (loading control). NPM and 18S band intensities were measured using ImageQuant and the stability of NPM mRNA was determined relative to the 18S for each time point. NPM signal in each one of these time points was compared with NPM level at 0 h of ActD treatment, which is considered 100%. These percentages were then plotted±s.e.m of three independent experiments. (c) C2C12 cells transfected with siRNA HuR or siRNA Ctr were fixed, permeabilized and incubated with digoxigenin-labelled in vitro-transcribed antisense probe to detect NPM mRNA (a,e) and with sense RNA probe (i,m) as a control (Ctr probe). Immunofluorescence staining with anti-HuR antibody (b,f,j,n) and with DAPI was performed. A single representative field for each cell treatment is shown. Scale bar, 10 μm. (d) Forty-eight hours post transfection of exponentially growing C2C12 cells with siRNA HuR or Ctr, polysomes were fractionated through sucrose gradients (15–50% sucrose). Absorbance at wavelength 254 nm was measured to determine the profile of polysome distribution. Ten fractions were collected and divided into two groups: non-polysome (NP, fractions 1–4) and polysome (P, fractions 5–10, contain mRNAs engaged in translation) (left panel). RT-qPCR was performed on each fraction using specific primers for NPM and GAPDH mRNAs (right panel). NPM mRNA level was standardized against GAPDH mRNA in each fraction and plotted±s.e.m. of three independent experiments.