Supplemental Figure 1. Replication restart is significantly abrogated in Atr/- cells. (A) An assay to measure replication recovery in cells by flow cytometry. BrdU incorporation was used to label cells before APH or other small molecule treatments. Thus, BrdU serves as a tag for S phase cells, which are monitored subsequently for replication recovery by EdU labeling. (B) Quantification of replication restart by flow cytometry for the indicated cell lines after varying times of APH treatment. (C) Phospho-S139 H2AX detection in BrdU-positive cells up to 48 hours after APH removal. S phase cells in the lines indicated were labeled with BrdU prior to 6-hour APH treatment. After APH removal, the BrdU-positive cells were gated and detected for phospho-S139 H2AX and changes in DNA content by propidium iodide staining. Atr/- cells remain H2AX positive long after APH removal and do not progress through the cell cycle as determined by DNA content. These effects are in contrast to those observed in ATR-expressing Atrflox/- cells.
Supplemental Figure 2. A large variety of CDK1/2 and PLK1 inhibitors promote replication recovery in ATR-deficient cells. (A) Replication restart assessed by flow cytometry following treatment with structurally distinct CDK1/2 and PLK1 small molecule inhibitors. CDK1/2 and PLK1 inhibitors were added one hour prior to 6 hours of APH treatment, after BrdU tagging. Therefore, only cells already in S phase at the time of inhibitor treatment are monitored, and the effects of CDK1/2 inhibition on G1 to S phase transition are avoided. (B) Quantification of replication recovery by flow cytometry shown in A. Note that the ability of CDK1/2 inhibitors to promote restart directly correlates with the specificity of these small molecules to inhibit CDK1 activity over CDK2 activity.
Supplemental Figure 3. Chemical inhibition of ATR is recoverable by shRNA suppression of RNF4. (A) Replication recovery on a population basis in Atr/- and Atrflox/- cells treated with three doses of ATR inhibitor with and without shRNA mediated suppression of RNF4. These results demonstrate that inhibition of Atr at these doses is recoverable by subsequent RNF4 suppression. (B) Western blot analysis of and H2AX phos-139 following addition of ATR inhibitor. The increase in H2AX phosphorylation observed using high doses of ATR inhibitor were also suppressible by RNF4 reduction (Fig. 5C), but replication recovery with these higher doses was not always observed, consistent with dominant-negative effects of ATR kinase inhibition.
Supplemental Figure 4. RAD51 is required for the replication restart fostered by PLK1 inhibition. (A) Quantification of RAD51 shRNA-mediated knockdown by western blot detection of total cellular protein. To achieve a graded reduction in RAD51 in Atrflox/- cells two distinct shRNAs were expressed via lentiviruses (RAD51 sh1, lanes 2 and 3; RAD51 sh2, lanes 4 and 5) at two levels of expression. Cells were transduced at multiplicities of infection of 5 (lanes 2 and 4, 95% infected) and 15 (lanes 3 and 5, 99% infected). Cells were harvested 48 hours later and whole cells lysates were prepared. (B) Replication restart in cells subjected to PLK1 inhibition and prior RAD51 suppression. Cellular replication restart was quantified by flow cytometry. The cells indicated were treated with APH or APH and PLK1 inhibitor (BI 2536) for 6 hours before harvest. Graded reduction of RAD51 was achieved as described in A, with the highest reduction in expression (High) created through infection of Atrflox/- cells with RAD51 sh2 at an MOI of 15. The replication recovery mediated by inhibiting PLK1 is largely abrogated when RAD51 expression is repressed. (C) Quantification of replication recovery shown in B.
Supplemental Figure 5. ShRNA targeting of RNF4. SUMO interacting domains are shown in red italics, the RING domain of RNF4 is shown in blue italics, and the shRNAs used to target RNF4 in these studies are labeled and are shown in bold and underlined.
1