Figure 7.
Inhibitor of EYA2 Tyr phosphatase blocks spindle formation in GSCs.(A) Dose- response curves of different cells to EYA2 Tyr phosphatase inhibitor, MLS000544460. The half-maximal inhibitory concentration (IC50) of EYA2 Tyr phosphatase inhibitor in different cells is shown to the right. Data are presented as the mean ± SD. (B) Relative cell numbers after treating different cell models with EYA2 Tyr phosphatase inhibitor (MLS000544460). Data are presented as the mean ± SD. **, P < 0.01; ***, P < 0.001. (C) Immunofluorescence staining of α-tubulin in GSC 3565 treated with EYA2 Tyr phosphatase inhibitor (EYA2i; MLS000544460). α-tubulin was labeled as green, DAPI as blue. Scale bars represent 5 µm. (D) Quantification of cells with abnormal spindle in GSC 3565 after treatment with EYA2 Tyr phosphatase inhibitor (MLS000544460). Data are presented as the mean ± SD. The P values were calculated by one-way ANOVA with Tukey’s multiple comparisons. ***, P < 0.001. (E) Flow cytometry–based cell cycle analysis following PI staining in GSC 387 or GSC 3565 treated with either DMSO or EYA2 Tyr phosphatase inhibitor (MLS000544460) with indicated concentrations. (F) Morphologies of spindles in GSC 387 or GSC 3565 treated with EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. α-Tubulin is in red and DAPI in blue. Scale bars represent 5 µm. (G) Quantification of GSC 387 (left) or GSC 3565 (right) with abnormal spindles after treatment with EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. Data are presented as mean ± SD. The P values were calculated by one-way ANOVA with Tukey’s multiple comparisons. ***, P < 0.001. (H) Flow cytometry–based cell cycle analysis following PI staining in GSC 387 or GSC 3565 treated with either DMSO or EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. (I) EYA2 mRNA expression in GSCs, liver cancer cells (Hep3B and PLC/PRF/5), and breast cancer cells (MDA-MB-231) measured by real-time qPCR. Data are presented as the mean ± SD. ***, P < 0.001. (J) Quantification of cells having abnormal spindles in GSCs, breast cancer cells, and liver cancer cells after treatment with 20 µM of EYA2 Tyr phosphatase inhibitor for 24 h. Data are presented as mean ± SD. ***, P < 0.001. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons. (K) Statistics of EdU+ cell in GSCs, breast cancer cells, and liver cancer cells. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons. (L) PERICENTRIN and phospho-histone 3 (pH3) staining in GSC 387 treated by MLS000544460 or vehicle. PERICENTRIN is in green, pH3 in red, and DAPI in blue. Scale bars represent 10 µm. (M and N) Quantification of pH3+ cells (M) and the pH3+ cells having abnormal centrosome foci numbers (N) in GSC 387 treated with either MLS000544460 (indicated as MLS4460) or vehicle. Abnormal PERICENTRIN foci were defined as 1, 3, or >4 foci per GSC. Data are presented as mean ± SD. Statistical significance was determined by t test. *, P < 0.05; ***, P < 0.001. A–K are representatives of three independent experiments. L–N are representative figures of four independent experiments using either GSC 387 treated with 4460 (2×) or GSC 3565 and GSC 3691 treated with 9987 (1× each). 12 or 7 different 120× images were quantified in D and G, respectively. At least six 120× images were quantified in B, and seven 80× images were quantified in E.

Inhibitor of EYA2 Tyr phosphatase blocks spindle formation in GSCs. (A) Dose- response curves of different cells to EYA2 Tyr phosphatase inhibitor, MLS000544460. The half-maximal inhibitory concentration (IC50) of EYA2 Tyr phosphatase inhibitor in different cells is shown to the right. Data are presented as the mean ± SD. (B) Relative cell numbers after treating different cell models with EYA2 Tyr phosphatase inhibitor (MLS000544460). Data are presented as the mean ± SD. **, P < 0.01; ***, P < 0.001. (C) Immunofluorescence staining of α-tubulin in GSC 3565 treated with EYA2 Tyr phosphatase inhibitor (EYA2i; MLS000544460). α-tubulin was labeled as green, DAPI as blue. Scale bars represent 5 µm. (D) Quantification of cells with abnormal spindle in GSC 3565 after treatment with EYA2 Tyr phosphatase inhibitor (MLS000544460). Data are presented as the mean ± SD. The P values were calculated by one-way ANOVA with Tukey’s multiple comparisons. ***, P < 0.001. (E) Flow cytometry–based cell cycle analysis following PI staining in GSC 387 or GSC 3565 treated with either DMSO or EYA2 Tyr phosphatase inhibitor (MLS000544460) with indicated concentrations. (F) Morphologies of spindles in GSC 387 or GSC 3565 treated with EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. α-Tubulin is in red and DAPI in blue. Scale bars represent 5 µm. (G) Quantification of GSC 387 (left) or GSC 3565 (right) with abnormal spindles after treatment with EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. Data are presented as mean ± SD. The P values were calculated by one-way ANOVA with Tukey’s multiple comparisons. ***, P < 0.001. (H) Flow cytometry–based cell cycle analysis following PI staining in GSC 387 or GSC 3565 treated with either DMSO or EYA2 Tyr phosphatase inhibitor (benzbromarone) at indicated concentrations. (I) EYA2 mRNA expression in GSCs, liver cancer cells (Hep3B and PLC/PRF/5), and breast cancer cells (MDA-MB-231) measured by real-time qPCR. Data are presented as the mean ± SD. ***, P < 0.001. (J) Quantification of cells having abnormal spindles in GSCs, breast cancer cells, and liver cancer cells after treatment with 20 µM of EYA2 Tyr phosphatase inhibitor for 24 h. Data are presented as mean ± SD. ***, P < 0.001. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons. (K) Statistics of EdU+ cell in GSCs, breast cancer cells, and liver cancer cells. Data are presented as mean ± SD. Statistical significance was determined by one-way ANOVA with Tukey’s correction for multiple comparisons. (L) PERICENTRIN and phospho-histone 3 (pH3) staining in GSC 387 treated by MLS000544460 or vehicle. PERICENTRIN is in green, pH3 in red, and DAPI in blue. Scale bars represent 10 µm. (M and N) Quantification of pH3+ cells (M) and the pH3+ cells having abnormal centrosome foci numbers (N) in GSC 387 treated with either MLS000544460 (indicated as MLS4460) or vehicle. Abnormal PERICENTRIN foci were defined as 1, 3, or >4 foci per GSC. Data are presented as mean ± SD. Statistical significance was determined by t test. *, P < 0.05; ***, P < 0.001. A–K are representatives of three independent experiments. L–N are representative figures of four independent experiments using either GSC 387 treated with 4460 (2×) or GSC 3565 and GSC 3691 treated with 9987 (1× each). 12 or 7 different 120× images were quantified in D and G, respectively. At least six 120× images were quantified in B, and seven 80× images were quantified in E.

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