Fig 2: Schematics illustrate the identified signaling cascade triggered by SCH treatment. In TNBC cells, SCH treatment causes DNA damage (increased phosphorylation levels of H2AX, ATM, p53, ATR, and CHK1), which leads to cell-cycle arrest. At the same time, SCH treatment suppresses DNA repair (reduced RAD51 and FANCD2), and DNA repair failure finally leads to apoptosis.

Fig 3: MiR-185 increases telomere dysfunction via the ATR signaling pathway in cancer cells. (A) ATR protein levels and phosphorylated Chk1 levels in POT1 knockdown, miR-185 overexpression and POT1 rescue HTC75 cells. Total Chk1 was blotted as a loading control. (B) After treatment with the ATR kinase inhibitor VE-821, the POT1 knockdown, miR-185 overexpression and control HTC75 cells were examined by immunostaining using anti-?-H2AX antibody and fluorescence in situ hybridization using the TTAGGG telomere probe. (C) Quantification of the percentage of cells in (B) with more than 3 TIFs. Error bars indicate standard deviations (n=3). (D) Growth curve of HTC75 cells stably infected with viruses encoding empty vector (miR-Neg), shPOT1, miR-185, miR-185 plus GFP, or miR-185 plus POT1 at different time points. (E) Telomere restriction fragment (TRF) analysis showed the telomere length of HTC75 cells stably infected with the indicated viruses at the indicated population doublings (PD 0, PD 30, PD 60). (F) Quantification of telomere length in (E).

Fig 4: Chk1 phosphorylation by ATR in G1 phase occurs in the absence of RPA2 phosphorylation and is independent of transcription. (A) Immunoblots of synchronized U2OS irradiated in G1 phase with 2 Gy and/or treated with ETP-46464 for 1 h (+15 to +75 min following IR). (B) Immunoblots of synchronized U2OS following 2 Gy and/or inhibition of transcription or BER. Synchronized U2OS cells in G1 were treated for 1 h with an inhibitor of RNA polymerases II and III (a-amanitin) or of APE1 (NSC332395) before exposure to 2 Gy IR. Inhibitors were not taken off and cells were harvested 1 h after IR. 127 × 122 mm (300 × 300 DPI).

Fig 5: Treatment with Berzosertib inactivates ATR-Chk1 related pathways and reduces cell migration in metastatic osteosarcoma cells. (A, B) Western blot after 48 h of Berzosertib exposure shows decreased expression of pATR and increased expression of ?H2AX and cleaved PARP in MNNG/HOS (A) and 143B (B) osteosarcoma cells. (C) Relative migration distance of MNNG/HOS and 143B osteosarcoma cells at different time points (0 h, 6 h, 12 h, 24 h, and 48 h) after Berzosertib treatment (scale bar: 50 µm). (D) Cell migration distance of MNNG/HOS and 143B osteosarcoma cells after exposure to Berzosertib. **p < 0.01 compared with the cell only (untreated) group. The data are mean ± SD of experiments carried out in triplicate.ATR, ataxia-telangiectasia and Rad3 related protein kinase; pATR, phospho-ATR.