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. 2023 Jan 19;12:e78202. doi: 10.7554/eLife.78202

Figure 1. Rapid PCNT and microtubule reorganization upon G0 arrest increases with HSA21 dosage.

(A) Model figure showing the three main consequences of elevated PCNT in trisomy 21 (T21) and tetrasomy 21 (Q21) compared to disomy 21 (D21) cells: (1) PCNT nucleates more microtubules; (2) PCNT forms large protein aggregates on microtubules; and (3) PCNT nucleates microtubules further away from the centrosome. These changes occur predominantly in the pericentrosomal region defined as 1.2–5 µm from the centroid of the centrosome. (B) Representative confocal images from time course experiments of RPE1 D21, T21, and Q21 cells grown on coverslips and serum depleted for 0, 2, 4, 8, 24, and 48 hr. Cells were stained with DM1a to label microtubules (MTs) and PCNT. Arrows point to cilium labeled by DM1a staining. (C) Quantitation of PCNT intensities in a 5 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (D) Quantitation of microtubule intensities in a 5 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (E) Quantitation of ciliation frequency throughout the time course using DM1a as a marker for cilia. Graph shows mean ± SD. N=3. (F) Quantitation of ciliation frequency per hour calculated by subtracting the percentage of ciliated cells at the starting time point from the percentage of ciliated cells at the ending time point and dividing the result by the total number of hours. Graph shows mean ± SD. N=3. Two-tailed unpaired t-test.

Figure 1—source data 1. Values for biological and technical replicates for graphs in Figure 1 and Figure 1—figure supplement 1.
elife-78202-fig1-data1.xlsx (136.2KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. Rapid PCNT and microtubule reorganization upon G0 arrest increases with HSA21 dosage.

Figure 1—figure supplement 1.

(A–C) Quantitation of whole cell PCNT intensities for D21 (A), T21 (B), and Q21 (C) cells throughout the time course normalized to average at 0 hr. Graphs show mean ± SD. N=3. Mann-Whitney U test. (D) Quantitation of centrosomal PCNT intensities in a 0–1.2 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (E) Quantitation of pericentrosomal PCNT intensities in a 1.2–5 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (F) Quantitation of centrosomal microtubule intensities in a 0–1.2 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (G) Quantitation of pericentrosomal microtubule intensities in a 1.2–5 µm radial circle around the centrosome throughout the time course normalized to D21 average at 0 hr. Graph shows mean ± SD. N=3. (H) Percentage of cells with a primary cilium over a 7-day time course. Graph shows mean ± SD. N=3.