Figure 6.
Figure 6. Effects of Sds22 on sister kinetochore dis-tances and interactions between microtubules and kinetochores. (A–D) Histograms showing distribution of interkinetochore distances (A and C) and interkinetochore distances per cell (B and D). (A and B) Sister kinetochore distances after Sds22 depletion. Hec1–Hec1 distances of sister pairs were measured in HeLa cells fixed 48 h after transfection with control siRNA or Sds22-specific siRNA (C and D) Hec1–Hec1 distances of sister pairs in untransfected HeLa cells or stable cell lines expressing GFP alone or GFP-Sds22 (C103 and D103). Data from two independent experiments are shown. Total number of cells: control siRNA, 28; Sds22 RNAi, 33; normal HeLa, 37; HeLa GFP, 30; C103, 42; D103, 28. (B) Kolmogorov-Smirnov test of significance: control siRNA and Sds22-depleted cells, P < 6 × 10−5. (D) Kolmogorov-Smirnov test of significance: normal HeLa and HeLa GFP, P < 0.05; HeLa GFP and C103, P < 6 × 10−6; HeLa GFP and D103, P < 1 × 10−5; C103 and D103, P < 0.9. (E) Effect of Sds22 on the microtubule–kinetochore attachment. Fluorescence lifetime measurement of FRET between EGFP-Hec1 and mCherry-tubulin is shown. Fluorescence lifetime of EGFP was determined in HeLa cells 48 h after transfection with either EGFP-Hec1 (green bars) or cotransfection with EGFP-Hec1 and mCherry-tubulin together with either control (blue bars) or Sds22-specific (red bars) RNAi duplexes. (F) Recruitment of BubR1 in Sds22-depleted cells. HeLa cells were fixed and immunostained 48 h after transfection with control or Sds22-specific RNAi duplexes. Bars, 10 µm. (G) Effect of Sds22 depletion in localization of BubR1. HeLa cells in F that showed a defined plate with aligned chromosomes were subdivided into cells where all chromosomes had clearly aligned to the metaphase plate as opposed to cells with one or more unaligned chromosomes. Both categories were scored on whether all, individual, or no kinetochores at all stained positive for BubR1. Data from four independent experiments are shown. Total number of cells: control RNAi, 153; Sds22 RNAi, 99. P-values between control and Sds22 RNAi were calculated by χ2 test: aligned kinetochores, P = 0.0004; unaligned kinetochores, P = 0.1172. Error bars indicate mean ± SEM.

Effects of Sds22 on sister kinetochore dis-tances and interactions between microtubules and kinetochores. (A–D) Histograms showing distribution of interkinetochore distances (A and C) and interkinetochore distances per cell (B and D). (A and B) Sister kinetochore distances after Sds22 depletion. Hec1–Hec1 distances of sister pairs were measured in HeLa cells fixed 48 h after transfection with control siRNA or Sds22-specific siRNA (C and D) Hec1–Hec1 distances of sister pairs in untransfected HeLa cells or stable cell lines expressing GFP alone or GFP-Sds22 (C103 and D103). Data from two independent experiments are shown. Total number of cells: control siRNA, 28; Sds22 RNAi, 33; normal HeLa, 37; HeLa GFP, 30; C103, 42; D103, 28. (B) Kolmogorov-Smirnov test of significance: control siRNA and Sds22-depleted cells, P < 6 × 10−5. (D) Kolmogorov-Smirnov test of significance: normal HeLa and HeLa GFP, P < 0.05; HeLa GFP and C103, P < 6 × 10−6; HeLa GFP and D103, P < 1 × 10−5; C103 and D103, P < 0.9. (E) Effect of Sds22 on the microtubule–kinetochore attachment. Fluorescence lifetime measurement of FRET between EGFP-Hec1 and mCherry-tubulin is shown. Fluorescence lifetime of EGFP was determined in HeLa cells 48 h after transfection with either EGFP-Hec1 (green bars) or cotransfection with EGFP-Hec1 and mCherry-tubulin together with either control (blue bars) or Sds22-specific (red bars) RNAi duplexes. (F) Recruitment of BubR1 in Sds22-depleted cells. HeLa cells were fixed and immunostained 48 h after transfection with control or Sds22-specific RNAi duplexes. Bars, 10 µm. (G) Effect of Sds22 depletion in localization of BubR1. HeLa cells in F that showed a defined plate with aligned chromosomes were subdivided into cells where all chromosomes had clearly aligned to the metaphase plate as opposed to cells with one or more unaligned chromosomes. Both categories were scored on whether all, individual, or no kinetochores at all stained positive for BubR1. Data from four independent experiments are shown. Total number of cells: control RNAi, 153; Sds22 RNAi, 99. P-values between control and Sds22 RNAi were calculated by χ2 test: aligned kinetochores, P = 0.0004; unaligned kinetochores, P = 0.1172. Error bars indicate mean ± SEM.

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