Journal: Nature Communications

Article Title: Stat3 regulates centrosome clustering in cancer cells via Stathmin/PLK1

doi: 10.1038/ncomms15289

Figure Lengend Snippet: ( a ) Quantification of centrosome clustering in paclitaxel-treated BT-549 cells with or without Stattic ( n =6 biological replicates, ≥1,200 cells per data point). ( b ) Positive kinase inhibitor ‘hits' identified from a screen of 400 known kinase inhibitors. n =2 biological replicates, ≥400 cells per data point. Error bars (s.d.). PLK1 and aurora kinase A (AURKA) were selected as potential downstream effector candidates of Stat3–Stathmin. ( c ) Quantification of phosphorylated to total PLK1 and AURKA ratios in Stat3-inhibitor-treated BT-549 cells using In-Cell Western assays ( n =4 biological replicates). ( d ) Quantification of phospho-PLK1 to PLK1 ratios in BT-549 cells stably expressing Stat3C or Stat3-Y705F and treated with Stat3 siRNA, as determined using In-Cell Western assays ( n =8 biological replicates). ( e ) Western blot of phospho-PLK1 and PLK1 from lysates of BT-549 cells treated with Stattic or BBI-608. Tubulin is a loading control. ( f ) ELISA-based quantification of in vitro PLK1 activity. Quantification of PLK1 activity assay using increasing concentrations of PLK1 (left panel; n =4 biological replicates). Quantification of relative PLK1 activity using 1 mU PLK1 and increasing amounts of Stathmin (middle panel; n =4 biological replicates). Quantification of recombinant PLK1 activity in the presence of recombinant Stathmin, Stat3 and Stattic (right panel; n =4 biological replicates). 1 mU (milli unit)=1 nmole of phosphate incorporated min −1 mg −1 . ( g ) Quantification of the effects of low and high concentrations of the PLK1 inhibitor BI-2536 and PLK1 siRNA on centrosome clustering in mitotic BT-549 cells ( n =4 biological replicates, 80 cells per condition). ( h ) Western blot of PLK1 siRNA-treated BT-549 cells stained for PLK1 and GAPDH (loading control). ( i ) Immunofluorescence images of γ-tubulin (red), DNA (Hoechst, blue) and Myc-Tag (inset, green) in a mitotic BT-549 cell transiently overexpressing Myc-tagged PLK1 and treated with 1 μM Stattic (left panels). Arrows point to clustered supernumerary centrosomes. Scale bar, 5 μm. Quantification of the percentage of Myc-PLK1-expressing mitotic BT-549 cells with declustered centrosomes (right panel; n =4 biological replicates, 120 cells per condition). ( j ) Diagram of potential Stat3–Stathmin–PLK1 pathway. * P <0.05; ** P <0.01; *** P <0.001. Error bars represent s.e.m. Statistical significance was tested between the indicated groups with analysis of variance in all graphs.

Article Snippet: Primary antibodies for immunofluorescence microscopy were: rabbit anti-pericentrin (Abcam ab4448; 1:2,000), mouse anti-α-tubulin (Sigma Aldrich T9026; 1:1,000), mouse anti-γ-tubulin (Abcam ab11316; 1:200), mouse anti-PLK1 (pT210; BD Bioscience 558400; 1:1,000), rabbit anti-PLK1 (Novus Biologicals NB100-56651; 1:500), rabbit anti-Aurora A (pT288; Abcam ab18318; 1:500), mouse anti-Aurora A (Sigma Aldrich A1231; 1:200), mouse anti-Centrin-2 (Santa Cruz Biotechnology sc-293192; 1:100), rabbit anti-centrin-2 (Santa Cruz Biotechnology sc-27793; 1:100) and rabbit anti-Myc (Cell Signaling Technology 2276S; 1:500).

Techniques: In-Cell ELISA, Stable Transfection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro, Activity Assay, Recombinant, Staining, Immunofluorescence