Figure 9: The HuR/KSRP-mediated decay activity requires PARN and EXOSC5 to destabilize NPM mRNA and promote muscle fibre formation.

(a) IP experiments using the anti-PARN antibody were performed on total cell extracts prepared from proliferating C2C12 cells treated or not with RNase A. The precipitates were used for western blot analysis with anti-PARN and -HuR antibodies. The blots shown are representative of two independent experiments. (b) IP coupled to RT-qPCR experiments was performed using the PARN or the EXOSC5 antibodies on total extract from C2C12 cells treated with siHuR or siKSRP. The NPM mRNA in the immunoprecipitate was standardized and normalized as described in Fig. 7i,j. The PARN- or EXOSC5-associated NPM mRNA levels in siHuR- or siKSRP-treated C2C12 cells were plotted relative to siCtr conditions±s.e.m. of three independent experiments, *P<0.01 (t-test). (c–f) GFP, -HuR and -CP2 proteins were expressed in C2C12 cells treated or not with siRNA-PARN or -EXOSC5. Twenty-four hours later, protein extracts and total RNA were prepared. (c) Western blot analysis was performed using antibodies against GFP and α-tubulin (loading control). (d–f) RT-qPCR analysis was performed using primers specific for GAPDH as well as PARN (d), EXOSC5 (e) and NPM (f) mRNAs. The levels of PARN, EXOSC5 and NPM mRNAs were normalized against GAPDH mRNA level and were plotted relatively to the siRNA Ctr-treated and GFP-transfected cells±s.e.m. of three independent experiments, *P<0.01 (t-test). (g–j) Confluent C2C12 cells depleted of PARN or EXOSC5 were induced for differentiation for up to 4 days. (g) Phase contrast pictures showing the differentiation status of these cells at day (d) d0 (a–c), d2 (d–f) and d3 (g–i). Scale bar, 50 μm. (h) These cells, on day 4 of muscle cell differentiation, were also fixed and used for IF with anti-myoglobin antibody and DAPI staining. Images of a single representative field were shown. Scale bar, 10 μm. (i) The fusion index was determined as described in Methods. (j) Total extracts from these cells were prepared and used for western blot analysis with antibodies against My-HC, NPM and α-tubulin (loading control).