Figure 5.
Figure 5. JAK1 interacts and colocalizes with paxillin in invadosome rings and regulates their assembly. (A) The specific coimmunoprecipitation of pax-WT-Flag and JAK1-GFP, transfected in HEK 293 cells, showed the interaction of these two proteins. (B) Immunoprecipitation of endogenous JAK1 in MEF-SrcY527F cells is associated with a specific coimmunoprecipitation of paxillin in comparison to rabbit polyclonal IgG control. (C) Confocal imaging of MEF-SrcY527F cells expressing paxillin-GFP and JAK1-RFP showed their colocalization. (D) TIRF imaging of MEF-SrcY527F cells expressing RFP-cortactin and JAK1-YFP showed the localization of this kinase in invadosome (top). Confocal imaging of endogenous JAK1 (total JAK1 or phospho-JAK1) revealed its accumulation in invadosomes (bottom). (E) Treatment with 25 µm Jak inhibitor I (P6) induced a decrease in STAT3 phosphorylation (left) associated with a significant decrease in invadosome formation (middle; n = 3; 350 cells counted per condition). Representative images extracted from a time series of MEF-SrcY527F cells expressing LifeAct-GFP and treated with 25 µm Jak inhibitor I (right). (F) Western blot of cell lysates from MEF-SrcY527F cells treated with control or JAK1 siRNA (left). F-actin staining of cells treated by these different siRNAs shows that JAK1 silencing significantly reduces invadosome formation (middle), as quantified at the right (n = 4, 500 cells counted per conditions), and induces the formation of stress fibers (middle). ***, P < 0.001. (G) Representative images extracted from the time series of MEF-SrcY527F expressing LifeAct-GFP revealed long-lasting large rings (red arrowhead) after JAK1 silencing in comparison to dynamic invadosome (multicolored arrowheads) in control condition. (H) Western blot analysis revealed the increase in JAK1 phosphorylation in response to the expression of constitutively activated mutant of Src, Src Y527F. (I) Western blot analysis of MEF-SrcY527F pax−/− shHic-5 cells expressing Hic-5 or pax-WT-Flag increases JAK1 phosphorylation. All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (C) 3 µm; (D) 5 µm; (F and G) 10 µm.

JAK1 interacts and colocalizes with paxillin in invadosome rings and regulates their assembly. (A) The specific coimmunoprecipitation of pax-WT-Flag and JAK1-GFP, transfected in HEK 293 cells, showed the interaction of these two proteins. (B) Immunoprecipitation of endogenous JAK1 in MEF-SrcY527F cells is associated with a specific coimmunoprecipitation of paxillin in comparison to rabbit polyclonal IgG control. (C) Confocal imaging of MEF-SrcY527F cells expressing paxillin-GFP and JAK1-RFP showed their colocalization. (D) TIRF imaging of MEF-SrcY527F cells expressing RFP-cortactin and JAK1-YFP showed the localization of this kinase in invadosome (top). Confocal imaging of endogenous JAK1 (total JAK1 or phospho-JAK1) revealed its accumulation in invadosomes (bottom). (E) Treatment with 25 µm Jak inhibitor I (P6) induced a decrease in STAT3 phosphorylation (left) associated with a significant decrease in invadosome formation (middle; n = 3; 350 cells counted per condition). Representative images extracted from a time series of MEF-SrcY527F cells expressing LifeAct-GFP and treated with 25 µm Jak inhibitor I (right). (F) Western blot of cell lysates from MEF-SrcY527F cells treated with control or JAK1 siRNA (left). F-actin staining of cells treated by these different siRNAs shows that JAK1 silencing significantly reduces invadosome formation (middle), as quantified at the right (n = 4, 500 cells counted per conditions), and induces the formation of stress fibers (middle). ***, P < 0.001. (G) Representative images extracted from the time series of MEF-SrcY527F expressing LifeAct-GFP revealed long-lasting large rings (red arrowhead) after JAK1 silencing in comparison to dynamic invadosome (multicolored arrowheads) in control condition. (H) Western blot analysis revealed the increase in JAK1 phosphorylation in response to the expression of constitutively activated mutant of Src, Src Y527F. (I) Western blot analysis of MEF-SrcY527F pax−/− shHic-5 cells expressing Hic-5 or pax-WT-Flag increases JAK1 phosphorylation. All graphs presented as means ± SD. Differences with a probability level P < 0.05 were considered significant in one-way ANOVA. Bars: (C) 3 µm; (D) 5 µm; (F and G) 10 µm.

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