Figure 8. BRISC modulates the function of NuMA in spindle pole assembly. (A) Depletion of BRISC regulates the interaction of NuMA with dynein and importin-β. HeLa cells transfected with control, ABRO1, or BRCC36 siRNA for 72 h were collected, and protein samples were analyzed by Western blotting with the indicated antibodies. (B) Depletion of BRISC decreased the incorporation of NuMA into the spindle poles during mitosis. HeLa cells transfected with control or BRCC36 siRNA were treated as described in MT regrowth assay in Materials and methods, and immunostained for α-tubulin (red) and NuMA (green). (C) Quantitative analysis of cells in which NuMA did not completely incorporate into the spindle poles within 25 min after NOC release. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (D) Representative images of the K-fiber minus ends distribution of NuMA. Control, ABRO1, or BRCC36-silenced HeLa cells were arrested by treatment with STLC and immunostained for NuMA (green) and α-tubulin (red). DNA was stained with DAPI. In control cells, NuMA accumulated and was orderly arranged as a ring-like structure surrounding the spindle pole body, whereas in ABRO1/BRCC36-depleted cells, this kind of structure was significantly disrupted. (E) Quantification of cells with normal (ring-like) NuMA distribution at monopolar poles, as shown in D. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (F) Quantitative analysis of the diameter in the z axis for NuMA accumulated at monopolar spindles in 3D reconstructions. Z stacks comprising 8–17 0.5-µm sections were acquired, and deconvolution of 3D image stacks were performed using LAS-AF software (Leica). Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (G) BRCC36 siRNA-induced irregular distribution of NuMA at spindle poles was rescued by CFP-BRCC36res WT. HeLa cells were cotransfected with BRCC36 siRNA and CFP-BRCC36res WT or CFP-B36res QSQ, treated with STLC, and then examined as described in D. (F) Graph showing the quantification of cells with normal NuMA distribution at monopolar poles, as shown in G. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. Bars, 5 µm. Error bars show mean ± SD.
Figure 8.

BRISC modulates the function of NuMA in spindle pole assembly. (A) Depletion of BRISC regulates the interaction of NuMA with dynein and importin-β. HeLa cells transfected with control, ABRO1, or BRCC36 siRNA for 72 h were collected, and protein samples were analyzed by Western blotting with the indicated antibodies. (B) Depletion of BRISC decreased the incorporation of NuMA into the spindle poles during mitosis. HeLa cells transfected with control or BRCC36 siRNA were treated as described in MT regrowth assay in Materials and methods, and immunostained for α-tubulin (red) and NuMA (green). (C) Quantitative analysis of cells in which NuMA did not completely incorporate into the spindle poles within 25 min after NOC release. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (D) Representative images of the K-fiber minus ends distribution of NuMA. Control, ABRO1, or BRCC36-silenced HeLa cells were arrested by treatment with STLC and immunostained for NuMA (green) and α-tubulin (red). DNA was stained with DAPI. In control cells, NuMA accumulated and was orderly arranged as a ring-like structure surrounding the spindle pole body, whereas in ABRO1/BRCC36-depleted cells, this kind of structure was significantly disrupted. (E) Quantification of cells with normal (ring-like) NuMA distribution at monopolar poles, as shown in D. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (F) Quantitative analysis of the diameter in the z axis for NuMA accumulated at monopolar spindles in 3D reconstructions. Z stacks comprising 8–17 0.5-µm sections were acquired, and deconvolution of 3D image stacks were performed using LAS-AF software (Leica). Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. (G) BRCC36 siRNA-induced irregular distribution of NuMA at spindle poles was rescued by CFP-BRCC36res WT. HeLa cells were cotransfected with BRCC36 siRNA and CFP-BRCC36res WT or CFP-B36res QSQ, treated with STLC, and then examined as described in D. (F) Graph showing the quantification of cells with normal NuMA distribution at monopolar poles, as shown in G. Mean ± SD of three independent experiments. ***, P < 0.001; Student’s t test. Bars, 5 µm. Error bars show mean ± SD.

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