3 homology arm  (Addgene inc)

Journal: bioRxiv

Article Title: Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targeting of WRN and ATR

doi: 10.1101/2023.07.28.550976

Figure Lengend Snippet: (A-B) Immunoblotting depicting the rapid induction of pKAP1, a marker of DNA double strand breaks, by 0.5 μM dTAG-13 in RKO (A) and KM12 (B) clones expressing FKBP-WRN. By contrast, dTAG-13 did not induce pKAP1 in OVCAR8 clones expressing FKBP-WRN even after extended treatment. One of two independent experiments is shown. (C) END-seq showing the rapid appearance of DNA breakage at unstable expanded TA repeats in KM12 cells treated with 0.5 μM dTAG-13. (D) Upper panel, immunoblotting showing that 0.5 μM dTAG-13 (24 h) induced significantly lower levels of pKAP1 in RKO and KM12 cells pre-treated for 24 h with 5 μM Palbociclib (PLB). Lower panel, successful inhibition of S-phase entry by PLB was confirmed by FACS-based cell cycle analyses. One of three independent experiments is shown. (E) Immunoblotting demonstrating the lack of CHK1 (S317) and RPA2 (S33) phosphorylation in dTAG-treated RKO FKBP-WRN cells. CHK1/RPA2 phosphorylation was also undetectable in KM12 FKBP-WRN cells except at 24 h after dTAG-13 treatment. Where indicated, cells were treated with 3 mM hydroxyurea (HU) for 2 h, either alone or in combination with 0.5 μM dTAG-13 (2 h dTAG-13 pre-treatment followed by 2 h HU+dTAG-13). Note that HU induced robust CHK1/RPA phosphorylation in both RKO and KM12 independently of WRN. One of two independent experiments is shown. (F) FACS-based cell cycle analyses showing that dTAG-treated RKO and KM12 cells failed to slow down or halt DNA synthesis during the first S-phase after acute WRN degradation. Note that WRN-degraded cells eventually completed S-phase and activated the G2 checkpoint. (G) Representative confocal images depicting RAD51 foci (maximum intensity projection) in RKO FKBP-WRN cells treated or not with 0.5 μM dTAG-13 for 24 h. Cyclin A (CCNA) and EdU positivity were used to distinguish cells in S-phase from those in G2. Note that RAD51 foci appear to be larger in G2 cells (EdU-negative, CCNA-high), as compared to S-phase cells (EdU-positive, CCNA-low/intermediate). (H) Quantification of RAD51 number per nucleus as a function of cell cycle position. (I) Volumetric analysis of RAD51 foci as a function of cell cycle position. One of two independent experiment is shown.

Article Snippet: DNA encoding the 5’- and 3’-homology arms of WRN was obtained from Twist Biosciences and subsequently cloned into pGMC00018, along with the PCR product encoding Puromycin R -2A-3X-FLAG-FKBP12 or eGFP-2A-3X-FLAG-FKBP12, using isothermal assembly.

Techniques: Western Blot, Marker, Clone Assay, Expressing, Inhibition, Phospho-proteomics, DNA Synthesis

Journal: bioRxiv

Article Title: Comprehensive mapping of cell fates in microsatellite unstable cancer cells support dual targeting of WRN and ATR

doi: 10.1101/2023.07.28.550976

Figure Lengend Snippet: (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.

Article Snippet: DNA encoding the 5’- and 3’-homology arms of WRN was obtained from Twist Biosciences and subsequently cloned into pGMC00018, along with the PCR product encoding Puromycin R -2A-3X-FLAG-FKBP12 or eGFP-2A-3X-FLAG-FKBP12, using isothermal assembly.

Techniques: Live Cell Imaging, Cell Tracking Assay, Expressing