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. 2014 Aug 12;3:e26016. doi: 10.7554/eLife.03254

Figure 3. RNA Polymerase II (RNAPII)-dependent transcription is required for the recruitment of CENP-A and its chaperone HJURP onto the centromere at early G1 (eG1).

(A) eG1 synchronized HeLa cells were treated or not (NT) with α-amanitin (α-ama) for 2 hr before staining for centromeric proteins CENP-A or CENP-B (green). The DAPI raw image is shown for a representative cell (cyan). Three independent experiments were performed and in each, a minimum of 30 cells were analyzed per slide. Scale bar: 5 μm. (B) Signal intensity of CENP-A and CENP-B spots from (A) was quantified using ImageJ, and relative ratios of α-amanitin vs non-treated conditions were determined. Means ± SD from three independent experiments is represented on the graph. Quantification values are tabulated in Supplementary file 1. (C) Centromeric proteins CENP-A and CENP-B (red) were co-stained with HJURP (green) and RNAPII phosphorylated on serine 2 (RNAPIIS2P, green), respectively, on centromeric chromatin fibers prepared from eG1 synchronized cells treated or not with α-amanitin for 2 hr (cyan, DAPI). Three independent experiments were performed and in each, a minimum of five chromatin fibers were analyzed per slide (co-localization on the same chromatin fiber after α-amanitin compared to non-treated: between CENP-B and RNAPIIS2P = 9/15 vs 10/15; and between CENP-A and HJURP = 2/15 vs 9/15). Scale bar: 1 μm. Co-IF, co-immunofluorescence.

Figure 3.

Figure 3—figure supplement 1. Inhibition of RNA Polymerase II-dependent transcription at early G1 (eG1) does not affect CENP-A and HJURP protein levels.

Figure 3—figure supplement 1.

(A) eG1-synchronized HeLa cells were treated with α-amanitin for 2 hr before staining for centromeric protein CENP-B (green) and DAPI (cyan). The heterogeneity of the CENP-B signal on a picture is shown with magnification of one cell with high signal (1 and 2) and low signal (3 and 4). Scale bar: 5 μm. (B) eG1-synchronized cells were treated, or not, with α-amanitin (2 hr), before total protein extraction, and the expression levels of CENP-A and HJURP were quantified by Western blot. The graph represents the average of three biological replicates, the y-axis plots the ratio (±SD) of protein level after α-amanitin compared to the non-treated condition for CENP-A and HJURP Western blot, respectively. IF, immunofluorescence.