Skip to main content

Advertisement

Fig. 2 | BMC Biology

Fig. 2

From: CCDC74A/B are K-fiber crosslinkers required for chromosomal alignment

Fig. 2

CCDC74A/B are required for spindle formation and integrity. a HeLa cells were transfected with negative control (siNC), CCDC74A/B-siRNA (siCCDC74A/B), or siCCDC74A/B together with siRNA-resistant Flag-CCDC74B (Flag-resCCDC74B) for 60 h. Western blots of endogenous CCDC74A/B and exogenous Flag-resCCDC74B (+, present; −, not present). GAPDH was loading control. bd HeLa cells were transfected with siNC, siCCDC74A/B, or siCCDC74A/B together with empty vector (EV) or with Flag-resCCDC74B for 60 h. b Immunofluorescence of α-tubulin (green) and pericentrin (red). DNA was stained with DAPI (blue). Scale bar, 5 μm. c Statistical analysis of bipolar spindle length in cells from b (3 independent experiments). d Statistical analysis of fluorescence intensity of spindle microtubules (MT) in cells from b. The signal from siNC was normalized to 1.0 (3 independent experiments). e Immunofluorescence of α-tubulin (green) and pericentrin (red) in cells from the knockout HEK293T and wild-type (WT) cells. DNA was stained with DAPI (blue). Scale bar, 5 μm. f Statistical analysis of spindle lengths of cells from e (3 independent experiments). g Time-lapse images of HeLa cells co-transfected with GFP-tubulin and either siNC or siCCDC74A/B for 60 h. Numbers in each frame indicate the time (minutes) after nuclear envelope breakdown (NEBD). Arrows indicate abnormal spindle poles. Scale bar, 5 μm. h Time required for bipolar spindle formation in g. i Ratios of multipolar/normal mitotic cells in each group from g. 4/65, 4 cells with multipolar spindles in 65 cells transfected with negative control siRNA. 14/55, 14 cells with multipolar spindles in 55 cells transfected with CCDC74A/B siRNA. In c, d, and f, data are mean ± SEM (one-way ANOVA test, ***P < 0.001, **P < 0.01, *P < 0.05; n.s., not significant). In h, data are mean ± SEM (unpaired two-tailed Student’s t test, ***P < 0.001)

Back to article page