Fig 3. Induction and repair of DNA damage in WRN-degraded MSI-H cancer cells monitored by time-lapse microscopy.

(A) Schematics of the RKO triple reporter line used for live cell imaging. An example of a vehicle-treated cells is shown below in the CFP, RFP and YFP channels. (B) Automated cell tracking was performed as described in the Methods section. Example traces of a vehicle-treated reporter cell over time; different phases of a cell cycle are indicated. The birth of a cell at mitosis (M) coincides with a reduction in nuclear area (as defined by H2B-mTurquoise) as well as PIP sensor intensity. PIP intensity increases in G1 but is then abruptly lost as a cell enters S-phase. Finally, PIP expression returns in G2. 53BP1 foci formation is also shown for this vehicle-treated cell. (C) Formation of 53BP1 puncta as a function of time. Cells were first imaged for approximately 20 h without drug to establish baseline 53BP1 levels. Subsequent addition of dTAG-13 resulted in a rapid induction of 53BP1 puncta over the baseline, which steadily increased over time. One of two independent experiments is shown. (D) Example traces of four dTAG-treated reporter cells. The two cells on the left panels showed induction of 53BP1 puncta during the first S-phase after WRN degradation. The two cells on the right panels had primarily induced 53BP1 in the first G2 after WRN degradation. Note however that these two cells also displayed a smaller induction of 53BP1 in the preceding S-phase. (E) Quantification of 53BP1 puncta intensity during the first cell cycle following acute WRN degradation revealed that peak 53BP1 induction occur predominantly in S and G2. One of two independent experiments is shown. (F) Analyses of metaphase spreads showed that a majority of dTAG-treated RKO cells failed to completely repair DNA breaks incurred in S/G2 and carried residual damages into the first mitosis. One of two independent experiments is shown.