Anti γh2ax ser139

For immunofluorescence analyses, we plated the cells in a proper density in cell culture μCLEAR plates (Greiner) and we fixed them in 4% formaldehyde in PBS. We incubated the cells with 0.25% Triton in PBS followed by 5% BSA in PBS.
Tissue sections were fixed in 10% buffered formalin (Sigma) and embedded in paraffin. After deparaffinization and citrate antigen retrieval, we incubated the slides with 0.5% Triton in PBS and blocked them with 1% BSA and 10% Australian FBS (Genycell) in PBS.
The antibodies were applied overnight in antibody diluents with background reducing agents (Invitrogen).
Primary antibodies: anti‐Rap1 1:500 (BL735, Bethyl), anti‐γH2AX Ser139 1:500 (05‐636, Millipore), anti‐TRF1 1:500 (BED5, Bio‐Rad).
Images were obtained using a confocal ultraspectral microscope (Leica TCS‐SP5). Quantifications were performed with Definiens software.

Thirteen human gastric cancer cell lines were used in this study (Table S1) and all cultures maintained at 37 °C in the presence of 5% CO2. SNU5 (IMDM + 20% FBS); MKN45, NCI-N87, SNU16, HGC27, AGS, NUGC3, SNU1 and NUGC4 (RPMI 1640 + 10% FBS); KATOIII (IMDM + 20% FBS); MGC803 and BGC823 (DMEM + 10% FBS). Abemaciclib (#S5716) and JQ1(#S7110) were obtained from SelleckChem LLC (Houston,TX). Ki-67 Rabbit mAb (#15,580) was purchased from Abcam. HRP conjugated Goat Anti-Rabbit IgG (H + L) secondary antibody (Servicebio Cat#GB23303, RRID:AB_2811189) was purchased from Servicebio. Cell Titer-Glo® (#G7570) was obtained from Promega. Crystal violet was obtained from Sigma. Antibodies against p21 (#2947T), p53 (#2524S), Phospho-Rb (Ser807/811) (#8516T), 53BP1 (#88,439) were purchased from Cell Signaling Technology (Danvers, MA). Anti- γH2AX (Ser139) (#05-636) was purchased from Millipore.

Cells were seeded on 12 mm cover slips in 6-well plates and treated for 48 h. After the treatments, cells were washed in PBS and fixed in 4% paraformaldehyde for 15 min in an orbital shaker. Following permeabilization with 0.2% of Triton-×-100 for 10 min at 4ºC, cells were blocked in 5% BSA: PBS and immunostained with anti-γH2AX (Ser 139) (Millipore) for 1 h. After washing with PBS, FITC-conjugated anti-mouse antibody (eBioscience) was added to the slides for 1 h. Thereafter, the washing was carried out with tris-buffered saline with 0.1% Tween-20 detergent (TBS-T), 4 % formaldehyde for 15 min and 0.2 % Triton-×-100, samples were hybridized with Cy3-labelled telomere sequence-specific PNA probes by first denaturing at 80°C for 6 min and incubation for 2 h in the dark at room temperature. Following hybridization, the slides were washed in formamide and 10% Tween-20 for 5 min. Finally, the cells were stained with DAPI (Vectashield^®). Approximately 100 images were captured on a fluorescence microscope (Carl Zeiss Axioplan 2, Oberkochen, Germany) with triple filters for simultaneous observation of DAPI (blue), FITC (green) and Cy3 (red).