Figure 1. Quantification of... | Rockefeller University Press

Figure 1.

$Figure 1. Quantification of SNAP-macroH2A in S/G2 and G1 phases of the cell cycle. (A) Western blot analysis of endogenous histone H3 and macroH2A before and after S phase. The time line of synchronization and harvesting of HEK 293T cells is shown. Equal numbers of cells were synchronized at the G1/S phase border by a double thymidine block. The cells were harvested, pre-S, or released from thymidine block and synchronized before M phase with RO-3306 and harvested, post-S. Chromatin fractions were isolated for Western blotting, testing endogenous macroH2A1, histone H3, and phosphorylation of H3 at serine 10 (H3S10P) as a marker of mitosis, with hnRNP k/j as a loading control. We show that H3 increases during mitosis, as expected, but there is no concurrent increase in macroH2A. Error bars represent standard deviation from three independent experiments. (B) The time line of synchronization and labeling of cells at S/G2 and G1 phases for the analysis in C. To label newly incorporated histones in S/G2 phase, HEK 293T cells stably expressing SNAP-tagged H3 or macroH2A were synchronized at the G1/S phase border by double thymidine block. Cells were treated with SNAP–Oregon Green to label preexisting histones (green arrow), subsequently blocking nonlabeled proteins using the nonfluorescent SNAP-Block reagent. The cells were allowed to progress to the G2/M transition until they were blocked using RO-3306 (a CDK1/cyclin B1 and CDK1/cyclin A inhibitor), labeling newly incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). To label newly incorporated histones in G1 phase, mitotic cells were collected by shake-off following nocodazole treatment for 12 h and spread onto coverslips. After 2 h, cells were labeled with Oregon Green and treated with the blocking reagent. Cells were then allowed to progress to the G1/S transition, when they were synchronized by double thymidine block, then labeling newly incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). After being released from the first synchronization, the cells were also incubated with EdU until the second synchronization, allowing cells that had undergone DNA synthesis to be identified. (C) An example of images showing the detection of preexisting and newly synthesized SNAP-tagged histones in S/G2 or G1 phases. Bar = 10 µm. (D) Image analysis measurements of red and green nuclear signals, representing the ratio of newly incorporated to preexisting histones H3 and macroH2A in the S/G2 and in G1 phases. The error bars represent one standard deviation from the number of single cells that indicated on each dataset. The P values were determined using two-tailed unpaired t tests.$

Quantification of SNAP-macroH2A in S/G2 and G1 phases of the cell cycle. (A) Western blot analysis of endogenous histone H3 and macroH2A before and after S phase. The time line of synchronization and harvesting of HEK 293T cells is shown. Equal numbers of cells were synchronized at the G1/S phase border by a double thymidine block. The cells were harvested, pre-S, or released from thymidine block and synchronized before M phase with RO-3306 and harvested, post-S. Chromatin fractions were isolated for Western blotting, testing endogenous macroH2A1, histone H3, and phosphorylation of H3 at serine 10 (H3S10P) as a marker of mitosis, with hnRNP k/j as a loading control. We show that H3 increases during mitosis, as expected, but there is no concurrent increase in macroH2A. Error bars represent standard deviation from three independent experiments. (B) The time line of synchronization and labeling of cells at S/G2 and G1 phases for the analysis in C. To label newly incorporated histones in S/G2 phase, HEK 293T cells stably expressing SNAP-tagged H3 or macroH2A were synchronized at the G1/S phase border by double thymidine block. Cells were treated with SNAP–Oregon Green to label preexisting histones (green arrow), subsequently blocking nonlabeled proteins using the nonfluorescent SNAP-Block reagent. The cells were allowed to progress to the G2/M transition until they were blocked using RO-3306 (a CDK1/cyclin B1 and CDK1/cyclin A inhibitor), labeling newly incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). To label newly incorporated histones in G1 phase, mitotic cells were collected by shake-off following nocodazole treatment for 12 h and spread onto coverslips. After 2 h, cells were labeled with Oregon Green and treated with the blocking reagent. Cells were then allowed to progress to the G1/S transition, when they were synchronized by double thymidine block, then labeling newly incorporated SNAP-tagged histones with SNAP-TMR Star (red arrow). After being released from the first synchronization, the cells were also incubated with EdU until the second synchronization, allowing cells that had undergone DNA synthesis to be identified. (C) An example of images showing the detection of preexisting and newly synthesized SNAP-tagged histones in S/G2 or G1 phases. Bar = 10 µm. (D) Image analysis measurements of red and green nuclear signals, representing the ratio of newly incorporated to preexisting histones H3 and macroH2A in the S/G2 and in G1 phases. The error bars represent one standard deviation from the number of single cells that indicated on each dataset. The P values were determined using two-tailed unpaired t tests.

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