Figure 1.
Tripolins inhibit Aurora kinase activity in vitro.
(A) Chemical structure of Tripolin A and Tripolin B. (B) Graph showing IC50 values (in µM) of Tripolin A (red) and Tripolin B (green) in the presence of different ATP concentrations, using an in vitro kinase assay. (C) Differential Scanning Fluorimetry results for Aurora A in the presence and absence of the inhibitors. Blue curve determines the melting temperature of Aurora A alone (45°C), red in the presence of Tripolin A (47°C) and green in the presence of Tripolin B (53°C).
Table 1.
Selectivity of Tripolins against a panel of kinases.
Figure 2.
Tripolin A selectively inhibits Aurora A over Aurora B in cultured tumor cells.
(A) Representative immunofluorescence images of HeLa cells in metaphase treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pAurora T288 green, DNA blue. (Scale bars, 5 µm). (B) Fluorescence intensity (% percentage) of pAurora A T288 on centrosomes and total Aurora A on spindles were quantified in control metaphase cells or cells treated with Tripolin A or Tripolin B (n≥20 cells for each group, from at least two independent experiments). **: 0.001<p<0.01; ***: p<0.001; ns: p>0.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (C) Western Blot analysis for Aurora A, Aurora B and pHistone H3 Ser10 in Tripolin A and Tripolin B-treated mitotic cells. α-tubulin was used as a loading control. (D) Representative immunofluorescence images of bipolar metaphase HeLa cells treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 24 h. In the merged images pHistone H3 Ser10 is pseudocolored red, Aurora B green, DNA blue. (Scale bars, 5 µm).
Figure 3.
Tripolin A treatment results in spindle and centrosomal defects.
(A) Representative immunofluorescence images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 24 h, 100 nM MLN8237 for 24 h or Aurora A siRNAs. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bars, 5 µm). (B) Graph showing the percentage of normal, multipolar, misaligned, disorganized and monopolar figures in control mitotic cells (DMSO or control siRNAs) and mitotic cells treated with Tripolin A, MLN8237 or Aurora A siRNA (n = 300 cells for each group, from three independent experiments). (C) Western Blot analysis for Aurora A levels in Aurora A siRNA treated cells. α-tubulin was used as a loading control. (D) Images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 5 h and 24 h or Aurora A siRNA. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 µm). (E) Graph showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO or control siRNA) and mitotic cells treated with Tripolin A, or Aurora A siRNA (n = 150 cells for each group, from three independent experiments).
Figure 4.
Tripolin A alters pole-to-pole distance and MT stability in mitotic cells and influences interphase MT array.
(A) Maximum projections from z-stacks of a representative control cell and representative cells treated with Tripolin A. In the merged images α-tubulin is pseudocolored red; pericentrin is green, DNA is blue. Yellow arrows indicate interpolar distance. (B) Interpolar distances were measured based on pericentrin staining in HeLa cells (n≥100 cells for each group, from at least three independent experiments). ***: p<0.0001; (Student's t-test, two-tailed). Error bars indicate SD. (C) Longitudinal line scans of tubulin intensity from metaphase spindles of control and Tripolin A treated HeLa cells (n = 5 for each group). Intensities were normalized to the maximum value of the control curve, and spindle size was interpolated. Curves indicate mean values. (D) Representative immunofluorescence images of HeLa cells in interphase treated with DMSO, 100 nM MLN8237 for 1 h or 20 µM Tripolin A for 1 h and 24 h. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bar 10 µm). (E) Graph showing the percentages of interphase cells with altered MT array, classified in the indicated arbitrary categories in control cells (DMSO) and cells treated with MLN8237 or Tripolin A (n = 150 cells for each group, from three independent experiments).
Figure 5.
Inhibition of Aurora A alters the localization of HURP.
(A) Representative immunofluorescence images of metaphase cells treated with DMSO, 20 µM Tripolin A or 100 nM MLN8237 for 24 h, or TPX2 siRNAs. In the merged images α-tubulin is pseudocolored red, HURP green. (Scale bars, 5 µm). (B) Longitudinal line scans of HURP intensity from metaphase spindles of control and Tripolin A treated HeLa cells (n = 5 for each group). Intensities were normalized to maximum value within the same spindle, and spindle size was interpolated. Curves indicate mean values. (C) Fluorescence intensity (% percentage) of HURP quantified in control metaphase cells and cells treated with Tripolin A (n≥20 cells for each group, from at least two independent experiments). ns p>0.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (D) Western Blot analysis for TPX2, in TPX2 siRNAs treated cells. α-tubulin was used as a loading control.
Figure 6.
Chemical structure of Tripolin A and Tripolin B.