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

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© 2014, Quénet et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 7. — Chromatin fibers were prepared from shRNA^scram, shRNA^sat1, or shRNA^sat2-transfected cells synchronized at eG1. To visualize centromeric domains, co-IF was performed for CENP-B (red), CENP-C (green) and RNA Polymerase II (phosphorylated on serine 2, RNAPII^S2P, green), whereas CENP-A (red) and HJURP (red) antibodies were co-stained with a DNA FISH probe against centromeric α-satellite DNA repeats (green). The DAPI raw image is shown for a representative chromatin fiber (cyan). Three independent experiments were performed and in each, five chromatin fibers were analyzed per slide (co-localization on the same chromatin fiber in shRNA^sat1 or shRNA^sat2-transfected cells: between CENP-B and RNAPII^S2P = 9/15 and 8/15; CENP-B and CENP-C = 14/15 and 13/15; between CENP-A and centromeric α-satellite DNA = 3/15 and 1/15; between HJURP and centromeric α-satellite DNA = 1/15 and 0/15, respectively). Scale bar: 1 µm. FISH, fluorescence in situ hybridization; IF, immunofluorescence.

Figure 7—figure supplement 1. — Chromatin fibers were prepared from shRNA^scram, shRNA^sat1, or shRNA^sat2-transfected cells synchronized at eG1. To visualize centromeric domains, co-IF was performed for CENP-B (red), CENP-C (green) and RNA Polymerase II (phosphorylated on serine 2, RNAPII^S2P, green), whereas CENP-A (red) and HJURP (red) antibodies were co-stained with a DNA FISH probe against centromeric α-satellite DNA repeats (green). The DAPI raw image is shown for a representative chromatin fiber (cyan). Three independent experiments were performed and in each, five chromatin fibers were analyzed per slide (co-localization on the same chromatin fiber in shRNA^sat1 or shRNA^sat2-transfected cells: between CENP-B and RNAPII^S2P = 9/15 and 8/15; CENP-B and CENP-C = 14/15 and 13/15; between CENP-A and centromeric α-satellite DNA = 3/15 and 1/15; between HJURP and centromeric α-satellite DNA = 1/15 and 0/15, respectively). Scale bar: 1 µm. FISH, fluorescence in situ hybridization; IF, immunofluorescence.