(A) The addition of increasing concentration of ATRi in HeLa cells causes a significant decrease in replication tract length, as measured by the ratio of the CldU tract length (with ATRi) to the IdU tract length (without ATRi). (B, C) Upon treatment with 2μM VE822 concomitant with CldU, knockdown of MED12 (B) or LIAS (C) showed a restoration of normal fork speed, as measured by the ratio of the CldU tract length (with ATRi) to the IdU tract length (without ATRi). The other hits do not affect fork slowing induced by ATRi. In all panels, the median values are indicated for each sample, and the asterisks indicate statistical significance. At least 100 fibers were quantified. (D) Table summarizing the impact on ATRi-induced cellular outcomes, observed for the seven top screen hits which were validated. “Yes” indicates differences which were statistically significant. The cell lines and ATRi used are indicated.