Inhibition or depletion of Hsp90 relocalizes DYRK3 to splicing speckles and leads to the accumulation of aberrant SC35 mitotic bodies.Figure 3(A, HeLa cells were left untreated (control) or treated with GA (5 µM), 17AAG (5 µM) or VER (10 µM) for 8 hrs. Cells were then fixed and immunostained for the nuclear speckle marker SC35 and DYRK3. Automatic segmentation of nuclear speckles is based on SC35. An automated imaging assay was used to quantify the percentage of nuclear speckles highly enriched for DYRK3 (using a fluorescent ratio 1.5 as threshold). Scale bar is 10 µm. (B) Number of speckles quantified = 1807 (control); 5110 (GA); 3596 (17AAG); 1556 (VER); p < 10-10, ± sem (One-way ANOVA). (C quantification of the percentage of mitotic cells showing aberrant SC35 mitotic bodies is reported (C). Number of cells counted: 923 (control), 694 (GA), 951 (17AAG) and 1019 (VER). n = 6 independent experiments, ± sem, p < 10-10, ± sem (One-way ANOVA). D) Representative confocal microscopy images of cells with SC35-positive bodies (D) Scale bar is 10 µm. (E) HeLa cells were lipofected with non-targeting control siRNA or siRNA pools specific for Hsp90 α and β. 72 hrs post-transfection, cells were fixed and stained for DYRK3 and SC35. Quantification of the percentage of mitotic cells showing aberrant SC35 mitotic bodies is reported. Number of cells counted: 644 (control); 654 (siRNA Hsp90 α and β). n = 3 independent experiments, ± sem; p = 0.01 (Student's t-test). (F) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3-dN. 24 hrs post-transfection GFP-DYRK3-dN cells were either left untreated or exposed to GA (5 µM) for 4 hrs. In untreated cells (control), GFP-DYRK3-dN is diffusely distributed in the cytosol and in the nucleus. Upon GA treatment, GFP-DYRK3-dN forms perinuclear (PN) aggregates. Representative confocal images are shown. Scale bar is 10 µm. (G) HeLa cells expressing GFP-DYRK3-dN were treated as described in F. GFP-DYRK3-dN PN aggregates were already visible 2 hrs after treatment with GA. GFP-DYRK3-dN mobility was investigated by FRAP in the cytoplasm and nucleus of untreated cells, and in the PN aggregates of GA-treated cells. The mean of 9-11 FRAP curves for each condition and the fitting curves are shown; sem is shown. (H) HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3 or GFP-DYRK3-dN. 24 hrs post-transfection cells were treated with sodium arsenite (A) to induce SGs and 45 min after they were allowed to recover for 4 hrs in drug-free medium (rec. control) or in presence of GA (5 µM; rec. GA). NP-40 soluble and insoluble proteins were fractionated and the distribution of GFP-DYRK3 and GFP-DYRK3-dN in both fractions was analyzed by immunoblotting. TUBA4A was used as loading control. Quantitation of the ration between GFP NP40 soluble and insoluble levels is shown; n = 3 independent experiments, ± sem (Student's t-test). (I) mCherry-G3BP1 expressing HeLa cells were lipofected with cDNAs encoding for GFP-DYRK3-dN or Sup35-NM-GFP-DYRK3-dN. 24 hrs post-transfection cells were treated for 45 min with sodium arsenite to induce SGs, followed by recovery for 4 hrs in presence of GA (5 µM; rec. GA). Representative confocal microscopy images are shown. Nucleic acid was stained with DAPI. Scale bar is 10 µm.