DNA2 and WRN are epistatic in stalled fork processing and replication restart. (A) Representative IdU tracts in RECQ1-, WRN-, or RECQ1/WRN-codepleted U-2 OS cells (out of 2 repeats; n ≥ 300 tracts scored for each dataset). (top) RECQ1 and WRN expression after shRNA knockdown. (B) Quantification of restarting forks in DNA2-, WRN-, or DNA2/WRN-codepleted cells. Mean shown, n = 3. Error bars, standard error. *, P < 0.05; **, P < 0.01 (paired t test). (top) Expression of WRN and DNA2 after shRNA knockdown. (C) Co-IP experiments in HEK293T cells transfected with empty vectors, Flag-DNA2, or Strep-HA-WRN. Cells were treated with 4 mM HU (3 h) where indicated. Whole-cell extracts were analyzed before (input) and after IP. (D) Statistical analysis of IdU tracts from RECQ1/DNA2-codepleted U-2 OS cells complemented with WT, ATPase-deficient (K671E), or nuclease-deficient (D294A) DNA2, when indicated. (E) Statistical analysis of IdU tracts from RECQ1-depleted WS cells complemented with WT, ATPase-deficient (K577M), or nuclease-deficient (E84A) WRN. Whiskers in D and E indicate the 10th and 90th percentiles. ns, not significant; ****, P < 0.0001 (Mann-Whitney test). n ≥ 300 tracts scored for each dataset shown in D and E.