Figure S2.
Characterisation of CENP-F-Mut1 and CENP-F-Mut2 cells.(a) Left: Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-FMut2 cells treated with 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and CENP-F(Ab90). Scale bar, 5 µm. Right: Quantification of kinetochore CENP-F intensity relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (b) Immunoblot of liquid N2 protein extracts from HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells treated with either control or CENP-F siRNA and arrested in nocodazole for 16 h. The membrane was probed with antibodies against CENP-F(Ab5) and α-tubulin. Asterisk indicates a nonspecific band. (c) Immunoblot of liquid N2 protein extracts from Rpe1 and Rpe1-Halo-CENP-F(KTD) cells arrested in nocodazole for 16 h. The membrane was probed with antibodies against CENP-F(Ab5) and α-tubulin. Asterisk indicates a nonspecific band. (d) Summary of CENP-F-Mut1 and CENP-F-Mut2 exon2 and exon19 sequencing. (e) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 3 wk and stained with DAPI and antibodies against CENP-C and CENP-F (Ab5). Scale bar, 5 µm. (f) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 5 wk and stained with DAPI and antibodies against CENP-C and CENP-F (Ab5). Scale bar, 5 µm. (g) Quantification of kinetochore CENP-F intensity relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 3 (left) or 5 (right) wk. (h) Immunoblot of liquid N2 protein extracts from HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells grown in culture for 3 or 5 wk and arrested in 330 nM nocodazole for 16 h. Membranes were probed with antibodies against CENP-F (Ab5) and α-tubulin. In a and g, boxes depict the median and first and third quartiles, and whiskers represent Q1 and Q3 ± 1.5× interquartile range. Asterisk indicates nonspecific bands.

Characterisation of CENP-F-Mut1 and CENP-F-Mut2 cells . (a) Left: Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-FMut2 cells treated with 330 nM nocodazole for 16 h and stained with DAPI and antibodies against CENP-C and CENP-F(Ab90). Scale bar, 5 µm. Right: Quantification of kinetochore CENP-F intensity relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells arrested in 330 nM nocodazole for 16 h. (b) Immunoblot of liquid N2 protein extracts from HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells treated with either control or CENP-F siRNA and arrested in nocodazole for 16 h. The membrane was probed with antibodies against CENP-F(Ab5) and α-tubulin. Asterisk indicates a nonspecific band. (c) Immunoblot of liquid N2 protein extracts from Rpe1 and Rpe1-Halo-CENP-F(KTD) cells arrested in nocodazole for 16 h. The membrane was probed with antibodies against CENP-F(Ab5) and α-tubulin. Asterisk indicates a nonspecific band. (d) Summary of CENP-F-Mut1 and CENP-F-Mut2 exon2 and exon19 sequencing. (e) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 3 wk and stained with DAPI and antibodies against CENP-C and CENP-F (Ab5). Scale bar, 5 µm. (f) Immunofluorescence microscopy images of HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 5 wk and stained with DAPI and antibodies against CENP-C and CENP-F (Ab5). Scale bar, 5 µm. (g) Quantification of kinetochore CENP-F intensity relative to CENP-C in HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells cultured for 3 (left) or 5 (right) wk. (h) Immunoblot of liquid N2 protein extracts from HeLa-K, CENP-F-Mut1, and CENP-F-Mut2 cells grown in culture for 3 or 5 wk and arrested in 330 nM nocodazole for 16 h. Membranes were probed with antibodies against CENP-F (Ab5) and α-tubulin. In a and g, boxes depict the median and first and third quartiles, and whiskers represent Q1 and Q3 ± 1.5× interquartile range. Asterisk indicates nonspecific bands.

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