Fig 2: ?H2AX staining and ATM/ATR pathway downregulation following MET suppression and enhancing sensitivity to olaparib. (A) Representative immunofluorescent staining of ?H2AX (red) and DAPI (blue) in DU145 and PC3 cells in the presence of Cri ((MET inhibitor crizotinib; 4 µM) and Ola (PARP inhibitor olaparib; 64 µM), Cri or Ola monotherapy, or dimethyl sulphoxide for 3 days. Scale bar, 50 µm. More than five foci per nucleus were considered as positive cells. (B) Western blot analysis of ?H2AX expression in DU145 and PC3 cells. (C-D) Western blot analysis of p-ATM/ATM, p-ATR/ATR, and RAD51 expression in DU145 and PC3 cells. Statistically significant differences were assessed by Student's t test in three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. ns = no statistical difference

Fig 3: Injected STEMIN and YAP5SA mmRNA induced cell replication and DNA replisome factors. (A) In a second injected heart, anti-phospho-histone H3 stain of a serial section was observed along two needle tracts marked by white arrows. Robust EdU stain overlap anti-pHH3 stains exactly in the same orientation and location. A key component of the DNA replisome was stained by anti-CLASPIN overlapped anti-pHH3 and Edu stains. Anti-NANOG stained NANOG was observed, but peaked levels were probably about 12 h earlier[15]. (B) In a third injected heart, anti-ORC2 (Origin recognition complex subunit 2) and anti-MCM2 showed the stained markers of the pre-initiation phase in early G1 stage induced in the injected hearts. Anti-ATR stained Ataxia telangiectasia and Rad3-related, an essential kinase that is active in S phase which overlapped other DNA replisome factors. Thus, STEMIN and YAP5SA mmRNA treatment fostered cell cycle entry by promoting DNA replication in the G1 phase.

Fig 4: Combined inactivation of CTPS and ATR is synthetically lethal to MYC-transformed cells. A, Apoptosis analysis of ARPE-19-MYC (DOX+) and ARPE-19-MYC (DOX-) cells treated with DAU and BAY-1895344 alone or together for 24 hours. B, Apoptosis analysis of ARPE-19-MYC (DOX+) cells treated with DAU alone or together with AZD6738 or VE-822 for 24 hours. C, The impacts of Q-VD-Oph on the combination of DAU and ATR inhibitor-induced apoptosis in ARPE-19-MYC (DOX+) cells. #, P < 0.001. D, Apoptosis analysis of ARPE-19-MYC (doxycycline+) cells treated with DAU (20 µmol/L)/BAY-1895344 (50 nmol/L) with or without 50 µmol/L cytidine (cyti), uridine (urid), guanosine (guan), or adenosine (aden) for 24 hours. E, Cell death analysis of ARPE-19-MYC (doxycycline+) and ARPE-19-MYC (doxycycline-) cells treated with DAU (20 µmol/L) and BAY-1895344 (50 nmol/L) alone or together for the indicated time. F, Apoptosis analysis of the indicated cells treated with DAU and BAY-1895344 alone or together for 48 hours. G, HCT116 and RKO cells expressing control or ATR shRNA were treated with DAU for 48 hours, followed by apoptosis analysis. *, P < 0.01; #, P < 0.001. H, Effect of DAU and BAY-1895344 on clonogenic survival of SW480 and RKO cells. I, The impacts of Q-VD-Oph on the combination of DAU- and BAY-1895344–induced apoptosis in the indicated cells. J and K, Control and MYC knockdown HCT116 cells were treated with DAU and BAY-1895344 for 48 hours, followed by Annexin V/PI–based apoptosis analysis (J) and immunoblotting analysis (K). Graphical data are means ± SEM. n = 3–4. For A, B, F, and J, *, P < 0.05; #, P < 0.0001; significantly different from DMSO-treated group. For D and I, *, P < 0.01; #, P < 0.0001; significantly different from DAU/BAY-1895344–treated group. DOX, doxycycline; ctrl, control; Cl. casp-3; cleaved caspase-3.

Fig 5: ATR/Chk1 activity is required for UV or MMS-induced autophagy. a and b ATR but not ATM activity is required for UV or MMS-induced LC3 aggregation. HeLa cells with stable expression of mRFP-LC3 pretreated 0.5 h with or without ATM inhibitor CP-466722 (10 µM) or ATR inhibitor VE-821 (1 µM) were subjected to fluorescence microscopy 4 h after UV (80 Jm-2) or 6 h after MMS (0.5 mM) treatment (a). The numbers of red puncta per cell were quantified using Imaris x64 image analysis software. Five random areas were counted for each experiment and data are presented as mean ± SD of three individual experiments. One-way ANOVA (F(8,126) = 165.07) followed by LSD post hoc test for multiple comparisons (b). c and d Chk1 but not Chk2 activity is required for UV or MMS-induced autophagy. HeLa cells with stable expression of mRFP-LC3 pretreated 0.5 h with or without Chk1 inhibitor (0.2 µM) or Chk2 inhibitor-II (4 µM) were treated and subjected to fluorescence microscopy as in panel a. Quantification of red puncta per cell was as in panel b. One-way ANOVA (F(8,126) = 229.17) followed by LSD post hoc test for multiple comparisons. e Treatment with Chk1 inhibitor attenuates UV or MMS-induced augment of LC3-II. HeLa cells pretreated 0.5 h with or without Chk1 inhibitor (0.2 µM) were subjected to immunoblotting assay 4 h after treated with UV (80 Jm-2) or 6 h after treated with MMS (0.5 mM). f Knockdown of Chk1 decreases the autophagic flux promoted by UV or MMS treatment. HeLa cells with stable expression of mRFP-GFP-LC3 and control shRNA (sh-Con) or shRNA against Chk1 (sh-Chk1-1 or sh-Chk1-2) were subjected to fluorescence microscopy 8 h after treated with UV (80 Jm-2) or 10 h after treated with MMS (0.5 mM). Scale bar, 10 µm