Figure S3.
(Related toFig. 5). (A) Schematic representation of experimental scheme used for CENP-HIKM RNAi. (B) Representative images of localization of CENP-A and CENP-HK after depletion of the CENP-HIKM complex in RPE-1 cells. CENP-HIKM RNAi was performed for 72 h using oligos for each subunit at 30 nM concentration. Cells were treated with STLC (5 µM) for 16 h to obtain a mitotic population before fixation. CENP-C was used to visualize kinetochores, and DAPI to stain DNA. Three biological replicates were performed. Scale bar: 5 μm. (C) Scatter plot of CENP-A levels at kinetochores for the experiment shown in B. n is the number of individually measured kinetochores. (D) Scatter plot of CENP-HK levels at kinetochores for the experiment in B. n is the number of individually measured kinetochores. (E) Representative images of localization of CENP-U after depletion of the CENP-HIKM complex in RPE-1 cells. CENP-HIKM RNAi was performed for 72 h using oligos for each subunit at 30 nM concentration. To obtain mitotic cells, cells were treated with STLC (5 µM) for 16 h before fixation. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (F) Scatter plot of CENP-U levels at kinetochores of the experiment shown in E. n refers to individually measured kinetochores. (G) Schematic representation of the experimental scheme used for CENP-T RNAi. (H) Representative images of localization of CENP-A and CENP-HK after depletion of the CENP-T complex in RPE-1 cells. CENP-T RNAi was performed for 60 h using oligos for each subunit at 30 nM concentration. Cells were treated with nocodazole (3.3 µM) for 4 h before fixation to enrich for mitotic cells. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (I) Scatter plot of CENP-A levels at kinetochores for the experiment in H. n is the number of individually measured kinetochores. (J) Scatter plot of CENP-HK levels at kinetochores for the experiment in H. n is the number of individually measured kinetochores. (K) Representative images of localization of CENP-O after depletion of the CENP-T complex in RPE-1 cells. CENP-T RNAi was performed for 60 h using oligos for each subunit at 30 nM concentration. Cells were treated with nocodazole (3.3 µM) for 4 h before fixation to obtain mitotic population. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (L) Scatter plot of CENP-O levels at kinetochores for the experiment in K. n is the number of individually measured kinetochores. Statistical analysis was performed with a nonparametric t test comparing two unpaired groups (Mann–Whitney test). Symbols indicate n.s.P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Red bars represent the median and interquartile range.

(Related to Fig. 5 ). (A) Schematic representation of experimental scheme used for CENP-HIKM RNAi. (B) Representative images of localization of CENP-A and CENP-HK after depletion of the CENP-HIKM complex in RPE-1 cells. CENP-HIKM RNAi was performed for 72 h using oligos for each subunit at 30 nM concentration. Cells were treated with STLC (5 µM) for 16 h to obtain a mitotic population before fixation. CENP-C was used to visualize kinetochores, and DAPI to stain DNA. Three biological replicates were performed. Scale bar: 5 μm. (C) Scatter plot of CENP-A levels at kinetochores for the experiment shown in B. n is the number of individually measured kinetochores. (D) Scatter plot of CENP-HK levels at kinetochores for the experiment in B. n is the number of individually measured kinetochores. (E) Representative images of localization of CENP-U after depletion of the CENP-HIKM complex in RPE-1 cells. CENP-HIKM RNAi was performed for 72 h using oligos for each subunit at 30 nM concentration. To obtain mitotic cells, cells were treated with STLC (5 µM) for 16 h before fixation. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (F) Scatter plot of CENP-U levels at kinetochores of the experiment shown in E. n refers to individually measured kinetochores. (G) Schematic representation of the experimental scheme used for CENP-T RNAi. (H) Representative images of localization of CENP-A and CENP-HK after depletion of the CENP-T complex in RPE-1 cells. CENP-T RNAi was performed for 60 h using oligos for each subunit at 30 nM concentration. Cells were treated with nocodazole (3.3 µM) for 4 h before fixation to enrich for mitotic cells. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (I) Scatter plot of CENP-A levels at kinetochores for the experiment in H. n is the number of individually measured kinetochores. (J) Scatter plot of CENP-HK levels at kinetochores for the experiment in H. n is the number of individually measured kinetochores. (K) Representative images of localization of CENP-O after depletion of the CENP-T complex in RPE-1 cells. CENP-T RNAi was performed for 60 h using oligos for each subunit at 30 nM concentration. Cells were treated with nocodazole (3.3 µM) for 4 h before fixation to obtain mitotic population. CENP-C visualizes kinetochores, and DAPI stains DNA. Three biological replicates were performed. Scale bar: 5 μm. (L) Scatter plot of CENP-O levels at kinetochores for the experiment in K. n is the number of individually measured kinetochores. Statistical analysis was performed with a nonparametric t test comparing two unpaired groups (Mann–Whitney test). Symbols indicate n.s.P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Red bars represent the median and interquartile range.

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