Figure S1.
NuSAP is not necessary for acentrosomal MT nucleation and is required for kinetochore–microtubule attachment. (A) Control and NuSAP-knockout HeLa cells were treated with 500 ng/ml nocodazole for 2 h. Then, the treated cells were released into a medium containing 15 ng/ml nocodazole and fixed at the indicated time. The cells were stained with anti-α-tubulin (green) and anti-γ-tubulin (red) antibodies. DNA was stained with DAPI (blue). (B) Quantification of the number of small acentrosomal MT asters when cells were released at 2 min (shown in A). Error bars indicate SD. 150 cells were quantified in three independent experiments (n = 3). Unpaired two-tailed t test. P = 0.9233. (C) Detection of the decay rate of PAGFP-α-tubulin signals over time in control and NuSAP KO cells. Three independent replicates of 14 cells per replicate were quantified (n = 3). (D) Control and NuSAP-knockout HeLa cells were placed on ice for 10 min before fixation. Then, the cells were processed for immunofluorescence using anti-Crest (red) and anti-Mad2 (green) antibodies. DNA was stained with DAPI (blue). The box areas were zoomed at the right. Scale bar in zoomed image, 1 μm. (E) Quantification of the percentage of kinetochores with Mad2 signal. Error bars indicate SD. Unpaired two-tailed t test. P = 0.0003. 338 and 340 kinetochores in 100 cells were counted from three independent experiments (n = 3). (F) Control and NuSAP-knockout HeLa cells synchronized to metaphase with MG132 and control HeLa cells synchronized to prometaphase with nocodazole release were stained with anti-α-tubulin (green) and anti-Crest (red) antibodies. DNA was stained with DAPI (blue). The box areas were zoomed at the right. Scale bar in zoomed image, 1 μm. (G) Measurement of interkinetochore (labeled by Crest) distances. Error bars indicate SD. P = 0.0002 for Ctrl/NuSAP KO; P = 0.7585 for NuSAP KO/Prometaphase. Three independent replicates of 100 pairs of kinetochores in 30 cells per replicate were quantified. Unpaired two-tailed t test. ns, not significant; ***, P < 0.001. n = the number of independent experiments presented. Scale bars, 10 μm unless specified otherwise.

NuSAP is not necessary for acentrosomal MT nucleation and is required for kinetochore–microtubule attachment. (A) Control and NuSAP-knockout HeLa cells were treated with 500 ng/ml nocodazole for 2 h. Then, the treated cells were released into a medium containing 15 ng/ml nocodazole and fixed at the indicated time. The cells were stained with anti-α-tubulin (green) and anti-γ-tubulin (red) antibodies. DNA was stained with DAPI (blue). (B) Quantification of the number of small acentrosomal MT asters when cells were released at 2 min (shown in A). Error bars indicate SD. 150 cells were quantified in three independent experiments (n = 3). Unpaired two-tailed t test. P = 0.9233. (C) Detection of the decay rate of PAGFP-α-tubulin signals over time in control and NuSAP KO cells. Three independent replicates of 14 cells per replicate were quantified (n = 3). (D) Control and NuSAP-knockout HeLa cells were placed on ice for 10 min before fixation. Then, the cells were processed for immunofluorescence using anti-Crest (red) and anti-Mad2 (green) antibodies. DNA was stained with DAPI (blue). The box areas were zoomed at the right. Scale bar in zoomed image, 1 μm. (E) Quantification of the percentage of kinetochores with Mad2 signal. Error bars indicate SD. Unpaired two-tailed t test. P = 0.0003. 338 and 340 kinetochores in 100 cells were counted from three independent experiments (n = 3). (F) Control and NuSAP-knockout HeLa cells synchronized to metaphase with MG132 and control HeLa cells synchronized to prometaphase with nocodazole release were stained with anti-α-tubulin (green) and anti-Crest (red) antibodies. DNA was stained with DAPI (blue). The box areas were zoomed at the right. Scale bar in zoomed image, 1 μm. (G) Measurement of interkinetochore (labeled by Crest) distances. Error bars indicate SD. P = 0.0002 for Ctrl/NuSAP KO; P = 0.7585 for NuSAP KO/Prometaphase. Three independent replicates of 100 pairs of kinetochores in 30 cells per replicate were quantified. Unpaired two-tailed t test. ns, not significant; ***, P < 0.001. n = the number of independent experiments presented. Scale bars, 10 μm unless specified otherwise.

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