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. 2015 Mar 18;14(10):1568–1582. doi: 10.1080/15384101.2015.1026489

Figure 4.

Figure 4

(See previous page). Large tumor suppressor (Lats)1/2 and CHO1 regulate the centrosomal localization and activity of LIM-kinase (LIMK)1 during mitosis. (A) Centrosomal localization of LIMK1-pT508 in metaphase HeLa-S3 cells transfected with a control (GL2) or CHO1/MKLP1-specific siRNA and synchronized by a thymidine single block-and-release (10 h). Scale bar, 10 μm. (B, C) The signal intensities of LIMK1-pT508 (B) and γ-tubulin (C) on centrosomes in CHO1/MKLP1 knockdown cells during metaphase. Data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (D) Centrosomal localization of phosphorylated LIMK1-pT508 and γ-tubulin in metaphase HeLa-S3 cells transfected with a control (GL2), Lats1-or Lats2-specific siRNA. Lats2 knockout cells (KO/HeLa-S3) were also transfected with Lats1 siRNA. The cells were synchronized by a thymidine single block-and-release (10 h). Scale bar, 10 μm. (E, F) The signal intensities of LIMK1-pT508 (E) and γ-tubulin (F) on the centrosomes in Lats1-, Lats2-, and Lats1/2-depleted cells during metaphase. Data represent the mean ± SD of n = 3 independent experiments (30 cells per experiment). (G) Immunoblot analyses of Cofilin-pS3 in HeLa-S3 cells transfected with a control (GL2) or CHO1/MKLP1-specific siRNA and synchronized by a thymidine single block-and-release (10 h). The ratio of the amount of Cofilin-pS3 in CHO1/MKLP1 knockdown cells to that in control cells is shown. (H) Immunoblot analyses of Cofilin-pS3 in HeLa-S3 cells transfected with a control (GL2), Lats1-, or Lats2-specific siRNA and synchronized by a thymidine single block-and-release (10 h). (I) Immunoblot analyses of Cofilin-pS3 in asynchronous parental HeLa-S3 cells, Lats1 KO/HeLa-S3 cells, Lats2 KO/HeLa-S3 cells, and Lats2 KO/HeLa-S3 cells transfected with Lats1 siRNA.