Figure S2.
CENP-C contributes to CENP-A deposition in quiescent cells. (A) Volcano plot comparing mRNA abundances in quiescent and cycling RPE1 cells as measured by RNA sequencing. Positive controls, including certain cyclins, cyclin-dependent kinases, and other proliferation factors, are highlighted in green. FOXM1 is highlighted in magenta. A P value cutoff was imposed at P = 6.84E−305 for genes with P values of 0. Genes with low read counts (total counts <50) were excluded. (B) Graph showing the fold change in mRNA abundance in mouse 3T3 cells between cycling cells and cells in quiescence for 7 days for the indicated centromere component as quantified by qPCR. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (C) Graph showing myc mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Myc mRNA is known to be unstable and have a short half-life. Fold change is calculated for each ActD time point by dividing by untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (D) Graph showing the correlations in the gene expression profiles of various centromere protein genes and FoxM1 across samples from the Broad Cancer Cell Line Encyclopedia data set (Barretina et al., 2012). Correlations are ordered from largest to smallest and obtained from Thiru et al. (2014). (E) Schematic showing experimental design for HaloTag pulse-chase experiments. Unblocked CENP-A is shown in green, blocked CENP-A in gray, and other histones in yellow. More experimental details can be found in the methods section. (F) Graph showing CENP-C intensity at the centromeres after 7 days of RNAi treatment. Cells were fixed and stained with CENP-C antibody at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Three replicates were conducted. **** indicates P < 0.0001, using unpaired t test with Welch’s correction. n = 486, 432 for control and CENP-C RNAi conditions, respectively (G) Graph showing total CENP-A intensity at the centromeres after 7 days of RNAi treatment. Cells were fixed and stained with CENP-A antibody at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Three replicates were conducted. ns = not significant. n = 481, 447 for control and CENP-C RNAi conditions, respectively. P = 0.3528, using unpaired t test with Welch’s correction. (H) Graph showing new, unblocked CENP-A intensity at the centromeres 6 days after blocking preexisting CENP-A and after 7 days of RNAi treatment. Cells were fixed and stained JF646 at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Six replicates were conducted. * indicates P < 0.05. n = 966, 879 for control and CENP-C RNAi conditions, respectively. P = 0.0161, using unpaired t test with Welch’s correction. (I) Representative immunofluorescent images showing levels of new unblocked CENP-A and CENP-C at the end of the pulse-chase experiment described in S2E. Cells were treated with either control or CENP-C siRNA. Scale bar = 10 µm. Refer to the image caption for details. Panel A: Volcano plot comparing m R N A abundances in quiescent and cycling R P E 1 cells. The horizontal axis represents the log 2 fold change (quiescence/cycling), and the vertical axis represents the negative log 10 p-value. Positive controls and F O X M 1 are highlighted. Panel B: Bar graph showing the fold change in m R N A abundance in mouse 3 T 3 cells for indicated centromere components. The vertical axis represents normalized abundance, and bars indicate mean and standard deviation. Panel C: Line graph showing myc m R N A abundance over time after Actinomycin D treatment. The horizontal axis represents time in hours, and the vertical axis represents normalized abundance. Panel D: Bar graph showing correlations in gene expression profiles of centromere protein genes and Fox M 1 across cancer cell lines. The vertical axis represents gene expression correlation to Fox M 1. Panel E: Schematic illustrating the experimental design for Halo Tag pulse-chase experiments. Panel F: Bar graph showing C E N P-C intensity at centromeres after R N A i treatment. The vertical axis represents normalized intensity, and points indicate average intensity per replicate. Panel G: Bar graph showing total C E N P-A intensity at centromeres after R N A i treatment. The vertical axis represents normalized intensity, and points indicate average intensity per replicate. Panel H: Bar graph showing new C E N P-A intensity at centromeres after blocking pre-existing C E N P-A and R N A i treatment. The vertical axis represents normalized intensity, and points indicate average intensity per replicate. Panel I: Immunofluorescent images showing levels of new unblocked C E N P-A and C E N P-C after pulse-chase experiment. All data is approximate.

CENP-C contributes to CENP-A deposition in quiescent cells. (A) Volcano plot comparing mRNA abundances in quiescent and cycling RPE1 cells as measured by RNA sequencing. Positive controls, including certain cyclins, cyclin-dependent kinases, and other proliferation factors, are highlighted in green. FOXM1 is highlighted in magenta. A P value cutoff was imposed at P = 6.84E−305 for genes with P values of 0. Genes with low read counts (total counts <50) were excluded. (B) Graph showing the fold change in mRNA abundance in mouse 3T3 cells between cycling cells and cells in quiescence for 7 days for the indicated centromere component as quantified by qPCR. CT values were normalized to those of GAPDH before comparing quiescent and cycling values. Graph shows at least three biological replicates, with three technical replicates each for each centromere mRNA. Bars represent mean ± SD. (C) Graph showing myc mRNA abundance over time after addition of 5 µg/ml actinomycin D in cycling and quiescent cells. Myc mRNA is known to be unstable and have a short half-life. Fold change is calculated for each ActD time point by dividing by untreated (0 h) value for each respective condition (quiescent or cycling). CT values were normalized to those of GAPDH before comparing treated and untreated values. Graph shows three biological replicates, with three technical replicates each. Bars represent mean ± SD. (D) Graph showing the correlations in the gene expression profiles of various centromere protein genes and FoxM1 across samples from the Broad Cancer Cell Line Encyclopedia data set (Barretina et al., 2012). Correlations are ordered from largest to smallest and obtained from Thiru et al. (2014). (E) Schematic showing experimental design for HaloTag pulse-chase experiments. Unblocked CENP-A is shown in green, blocked CENP-A in gray, and other histones in yellow. More experimental details can be found in the methods section. (F) Graph showing CENP-C intensity at the centromeres after 7 days of RNAi treatment. Cells were fixed and stained with CENP-C antibody at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Three replicates were conducted. **** indicates P < 0.0001, using unpaired t test with Welch’s correction. n = 486, 432 for control and CENP-C RNAi conditions, respectively (G) Graph showing total CENP-A intensity at the centromeres after 7 days of RNAi treatment. Cells were fixed and stained with CENP-A antibody at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Three replicates were conducted. ns = not significant. n = 481, 447 for control and CENP-C RNAi conditions, respectively. P = 0.3528, using unpaired t test with Welch’s correction. (H) Graph showing new, unblocked CENP-A intensity at the centromeres 6 days after blocking preexisting CENP-A and after 7 days of RNAi treatment. Cells were fixed and stained JF646 at the end of the experiment from S2E. Each point indicates the average of centromere intensity level adjusted for background for each replicate. Intensity values were normalized to control. Six replicates were conducted. * indicates P < 0.05. n = 966, 879 for control and CENP-C RNAi conditions, respectively. P = 0.0161, using unpaired t test with Welch’s correction. (I) Representative immunofluorescent images showing levels of new unblocked CENP-A and CENP-C at the end of the pulse-chase experiment described in S2E. Cells were treated with either control or CENP-C siRNA. Scale bar = 10 µm.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal