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. 2019 Jan 1;33(1-2):75–89. doi: 10.1101/gad.315978.118

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© 2019 Moquin et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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Figure 7. — ZPET represses HR, slows replication forks under stress, and increases cellular sensitivity to CPT and olaparib. (A) Wild-type cells, ZPET knockout cells, and ZPET knockout cells reconstituted with HA-ZPET were analyzed for HR efficiency using the mClover assay. The left panel is a schema of the mClover assay. In the right panel, each data point represents an independent sample. Red and blue data points were generated using two subclones of reconstituted ZPET knockout cells (>900 cells were analyzed for each sample). (B) ZPET knockout cells reconstituted with Dox-inducible HA-ZPET were sequentially labeled with CIdU and IdU as indicated. Cells were treated with 250 µM HU or 2.5 µM CPT during IdU labeling. (Right panel) The length of replication tracts before HU/CPT treatments (CIdU-labeled) was analyzed. (Left and middle panels) The effects of HU/CPT on replication tracts (IdU/CIdU ratios) were analyzed. The length of 200 replication tracts were measured. n = 200. (Black bars) Mean length or mean ratio. Significance was determined by Mann-Whitney test. (****) P < 0.0001; (ns) not significant. (C) Wild-type and ZPET knockout U2OS lines were treated with increasing doses of CPT. Cell viability was analyzed in 7 d. Error bars represent SD. n = 3 technical triplicates. (D) ZPET knockout and reconstituted cells were treated with increasing doses of CPT. Cell viability was analyzed in 7 d. (E) Wild-type and ZPET knockout cells were treated with increasing concentrations of olaparib. Cell viability was analyzed in 7 d. (F) Wild-type and ZPET knockout U2OS cells were transfected with control or BRCA1 siRNA as indicated and treated with increasing doses of olaparib. Cell viability was analyzed in 5 d.