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. 2016 Aug 23;5:e17548. doi: 10.7554/eLife.17548

Figure 4. E2-induced R-loops occur on chromatin marked by DNA damage and RNase H reduces E2-induced DSBs.

(A) Proximity ligation assay between S9.6 antibody and P-H2AX antibody in cells treated either with 0 or 100 nM E2 for 24 hr. (B) Quantification of the percentage of cells with ≥ 1 PLA focus per nucleus. Single-antibody controls from cells treated with 100 nM E2 for 24 hr are shown. Error bars represent the SEM from 4 biological replicates. **p<0.01 (Student’s t-test). (C) P-H2AX and P-KAP1 levels from co-IP of S9.6 or IgG from cross-linked and sonicated cells treated with 100 nM E2 for 24 hr. Input is 60% of the IP. (D) Western blot with Flag antibody in MCF7 tetOn-RH cells. 1000 ng/mL doxycycline (DOX) was added for 48 hrs where indicated. (E) Immunostaining for P-H2AX and FLAG in MCF7 tetON-RH cells treated with increasing concentrations of DOX (100, 250, 500 1000 ng/mL) for 24 hrs prior to the addition of either 0 or 100 nM E2 for 24 hr. (F) Quantification of P-H2AX intensity for the experiment described in (E), where the triangle indicates increasing concentrations of DOX. ****p<0.0001 (non-parametric Mann-Whitney rank sum t-test). n = 3 biological replicates. >1000 cells/condition quantified. (G) Neutral comet assay in MCF7-tetOn-RH cells treated with or without 1000 ng/mL DOX for 24 hrs prior to 0 nM E2 or 100 nM for 24 hr. (H) Quantification of the neutral comet tail moment described in (G). **p<0.01 (non-parametric Mann-Whitney rank sum t-test). n = 3 biological replicates. >100 comets/condition. a.u. = arbitrary units.

DOI: http://dx.doi.org/10.7554/eLife.17548.022

Figure 4.

Figure 4—figure supplement 1. Quantification of the fold change in the percent of cells with >5 P-H2AX foci per cell in MCF7-tetON-RH cells treated with indicated concentrations of DOX. *p<0.05.

Figure 4—figure supplement 1.

Error bars represent SD of 3 technical replicates.
DOI: http://dx.doi.org/10.7554/eLife.17548.023
Figure 4—figure supplement 2. Expression of RNase H prevents E2-induced DNA damage.

Figure 4—figure supplement 2.

(A) Quantification of P-H2AX intensity per nucleus in MCF7 tetON-RH clone 6 (left) or MCF7 tetON-RH clone 8 (right) treated with 0 or 1000 ng/mL DOX for 24 hr prior to the addition of 0 nM or 100 nM E2 for 24 hr. ****p<0.0001 (non-parametric Mann-Whitney rank sum t-test). n = 3 technical replicates. >500 cells/condition. (B) Western blot with Flag antibody in MCF7 tetOn-RH cells, clones 6 and 8. DOX was added at 1000 ng/mL MCF7 tetOn-RH cells for 48 hr.
DOI: http://dx.doi.org/10.7554/eLife.17548.024
Figure 4—figure supplement 3. FACS profiles of MCF7 tetON-RH cells treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 100 nM E2 for 24 hr.

Figure 4—figure supplement 3.

Cells were pulsed with 25 μM BrdU for 30 min prior to fixation. DNA content is marked by propidium iodide (x-axis) and BrdU incorporation is shown on the y-axis. The percentage of cells in each of the four cell-cycle quadrants is shown.
DOI: http://dx.doi.org/10.7554/eLife.17548.025
Figure 4—figure supplement 4. EU incorporation following RNase H expression in MCF7 cells.

Figure 4—figure supplement 4.

(A) EU staining in MCF7 tetON-RH cells either treated with or without 1000 ng/mL DOX for 24 hr prior to the addition of 0 or 100 nM E2 for 24 hr (48 hr DOX total). 100 μM DRB was added for 2 hr. Cells were pulsed with 100 μM EU for 30 min before fixation. Hoechst is used to stain the nucleus. (B) Quantification of EU staining for the experiment described in (A). n = 2 biological replicates. >200 cells/condition. a.u. = arbitrary units.
DOI: http://dx.doi.org/10.7554/eLife.17548.026