Fig. 3

MPS1 silencing or inhibition reduces multiple centrosome presence in U2OS-E7 cells. A Silencing of MPS1 by shRNA. MPS1 protein levels in U2OS-E7 and control cells were determined by western blot assay. Also, U2OS-E7 stable expressing cells were transfected with four different short hairpins ribonucleic acid (shRNA) constructs against MPS1 (MPS1 shRNA), thus silencing the MPS1 gene. As a negative control, U2OS-E7 stable expressing cells were transfected with a random sequence (shRNA control), and no effect on MPS1 expression was observed. B Immunodetection of centrosomes in U2OS-E7 cells. Immunofluorescence was performed to detect γ-tubulin (red) and centrin (green), contrasting the nucleus with DAPI (blue). Representative images of mononuclear cells with centrosomal abnormalities (γ-tubulin signals and multiple centrin signals) in U2OS-E7 stable expressing cells (II), mononuclear cells with normal centrosome (two γ-tubulin and centrin signals) in U2OS-E7 stable expressing cells transfected with the four shRNA MPS1 constructs (III), and mononuclear cells with centrosomal abnormalities (multiple γ-tubulin and centrin signals) in U2OS-E7 stable expressing cells transfected with a random shRNA construct (IV) are shown. The objective used was 100×. C Quantification of the percentage of cells with multiple centrosomes treated with MPS1 siRNAs. Mononuclear cells with γ-tubulin and centrin signals were counted in three individual experiments. A statistically significant higher percentage (p < 0.01) was observed in U2OS-E7 stable expressing cells when compared to control cells. This amplification of centrosomes is significantly decreased by inhibiting MPS1 with shRNAs (p < 0.01). D Quantification of the percentage of cells with multiple centrosomes treated with MPS1-IN-3 inhibitor. Mononuclear cells with γ-tubulin and centrin signals were counted in three individual experiments. A statistically significant higher percentage (p < 0.001) was observed in U2OS-E7 stable expressing cells compared to control cells. This amplification of centrosomes is significantly decreased by inhibiting MPS1 kinase activity only with 50 µg MPS1-IN-3 (p < 0.01)