DYRK3 is a new client of Hsp90.Figure 2(A) Schematic model showing how Hsp90 could regulate DYRK3 stability and activity. (B) HeLa cells were lipofected with a cDNA encoding for an empty vector or GFP-DYRK3. Where indicated, cells were treated for 2 hrs with GA (5 µM). Cell lysates were subjected to immunoprecipitation of GFP-DYRK3. Levels of GFP-DYRK3 and endogenous Hsp90 are shown in the input and bead fractions. (C) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 368-504 cells counted/sample, p < 0.002 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 10-6 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GA (5 µM) (One-way ANOVA). Scale bar is 10 µm. (D) HeLa cells were lipofected with a cDNA encoding for an empty vector or GFP-DYRK3. Where indicated, cells were treated for 2 hrs with GSK (5 µM). Cell lysates were subjected to immunoprecipitation of GFP-DYRK3. Levels of GFP-DYRK3 and endogenous Hsp90 are shown in the input and bead fractions. (E) HeLa cells were subjected to proximity ligation assay (PLA) using antibodies specific for endogenous Hsp90 and DYRK3. PLA foci and nuclei were segmented, and PLA foci/cell were automatically quantified. The mean of PLA/foci in cells incubated with no antibodies (-), only Hsp90 antibody, only DYRK3 antibody and both Hsp90 and DYRK3 antibodies is shown. Cells incubated with Hsp90 and DYRK3 antibodies and left untreated were used as control. n = 3 independent experiments, ± sem; 287-578 cells counted/sample, p < 10-6 between the negative controls and cells incubated with Hsp90 and DYRK3 antibodies; p = 0.002 between cells incubated with Hsp90 and DYRK3 antibodies and left untreated (control) or treated for 2 hrs with GSK (5 µM) (One-way ANOVA). (F) HeLa cells were lipofected with a cDNA encoding for GFP or GFP-DYRK3. 6 hrs post-transfection, cells were incubated in drug-free medium (-) or in presence of GA, 17AAG or VER (concentrations are shown). Total proteins were extracted 16 hrs later. GFP and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments. (G) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 16 hrs post-transfection, cells were incubated for an additional 8 hrs in drug-free medium (-) or in presence of GA or 17AAG alone or combined with MG132 (concentrations are shown). Total proteins were extracted and GFP, ubiquitin and TUBA4A protein levels were analyzed by immunoblotting. Representative immunoblotting of 3 independent experiments. (H) HeLa cells were lipofected with non-targeting control siRNA (-) or siRNA pools specific for Hsp90 α and/or β. 48 hrs later, cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, total proteins were extracted and GFP-DYRK3, Hsp90 α and β, and TUBA4A protein levels were analyzed by immunoblotting. GFP-DYRK3 protein levels were quantified and are shown. n = 3 independent experiments, ± sem; p < 0.001 (Student's t-test). (I) HeLa cells were lipofected with a cDNA encoding for GFP-DYRK3. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM) alone (control) or with GA (5 µM) or 17AAG (5 µM). Cell were then fixed and the percentage of cells expressing GFP-DYRK3 with SGs was counted. Total cells counted: 462 (sodium arsenite); 444 (sodium arsenite and GA); 561 (sodium arsenite and 17AAG). n = 3 independent experiments, ± sem; p < 0.001 (One-way ANOVA). (J) HeLa cells were either left untreated or lipofected with a cDNA encoding for GFP. 24 hrs post-transfection, cells were treated for 45 min with arsenite (50 µM). The percentage of cells forming SGs is shown (only GFP-positive cells were counted in the GFP-overexpressing population). Total cells counted: 934 (control) and 555 (GFP). n = 3-4 independent experiments, ± sem (Student's t-test).