Fig. 4

The Aurora kinase A/Plk1 axis is hyper-activated in AdvSM and affects SETD2/H3K36Me3 expression. A Aurora A and Plk1 expression and phosphorylation in HMC-1 cells assessed by WB. B Representative Western blot results for Aurora A and Plk1 protein expression and phosphorylation in SM patients (4 ISM and 4 ASM) as compared to a pool of healthy donors (HDs). C Box plots of phospho-Aurora A and phospho-Plk1 [P-AKA(T288) and P-Plk1(T210)] expression levels estimated by densitometric analysis of Western blots in 11 ISM patients (grey) and 14 AdvSM patients (red). Signal intensities in single blots obtained from three individual experiments were quantified in comparison to the signal intensities obtained for the pool of HDs, used as control. D Co-immunoprecipitation assay showing that total and phospho-Aurora A can be found bound to serine/threonine phosphorylated SETD2 after bortezomib treatment in the SETD2-deficient HMC-1 cells. E and F Silencing or inhibiting Aurora A by siRNA (for 72 h) or by alisertib (500 nM for 24 h), respectively, induced SETD2 release from Aurora A and de-phosphorylation, and resulted in the rescue of SETD2 expression in HMC-1 cells. A negative control (NC) obtained by the immunoprecipitation of protein lysates using a resin conjugated with an anti-IgG1 antibody was loaded for each immunoprecipitation experiment