Figure 6: KSRP is required for the HuR-mediated destabilization of NPM mRNA.

(a,b) Exponentially growing C2C12 cells were treated with siRNA Ctr or siRNA against HuR or KSRP. Total cell (a) or RNA (b) extracts from these cells were prepared 48 h after transfection. (a) Western blot analysis was performed using antibodies to detect KSRP, NPM, HuR and α-tubulin (loading control). (b) RT-qPCR analysis was performed to assess NPM mRNA levels that were standardized against GAPDH mRNA. The levels of NPM protein (a) or mRNA (b) in cells depleted of HuR or KSRP were plotted relative to the siRNA Ctr condition±s.e.m. of three independent experiments. (c) The stability of the NPM, MyoD and p21 mRNA in C2C12 cells depleted or not of KSRP was determined by AcD pulse-chase experiments. RT-qPCR analysis was performed using specific primers for these three mRNAs. mRNA levels were then standardized against GAPDH mRNA and plotted±s.e.m of three independent experiments. (d,e) Exponentially growing C2C12 cells were transfected with the pcDNA3, pcDNA-Flag-KSRP, GFP and GFP-HuR. (d) Total cell extracts or (e) RNA extracts from these cells were prepared 24 h post transfection. (d) Western blot was performed using antibodies against Flag, HuR or α-tubulin (loading control). (e) RT-qPCR analysis was performed as described above in c and mRNA levels were plotted relative to their levels in control cells±s.e.m. of three independent experiments. (f–h) GFP and GFP-HuR plasmids were transfected in C2C12 cells depleted or not of KSRP. Twenty-four hours later total protein extracts and RNA were prepared. (f,g) Western blot analysis was performed with antibodies against (f) KSRP, GFP and (g) NPM. α-tubulin levels were assessed as a loading control in both f and g. (h) RT-qPCR analysis was performed as described above. NPM mRNA levels were standardized against GAPDH mRNA level. In each condition, the level of NPM mRNA in siKSRP-treated cells was plotted relative to its levels in siRNA Ctr-treated and GFP-transfected cells±s.e.m. of three independent experiments. In the histograms presented in a,b,e,h, *P<0.01, **P<0.001, ***P<0.0001 (t-test).