Dna pk

Antibodies for western blotting were purchased from Cell Signaling (Danvers, MA): p-EGFR (Tyr1068) (2234, 1:1000), EGFR (4267, 1:1000), p-MEK1/2 (Ser217/221) (9154, 1:1000), MEK1/2 (8727, 1:1000), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (9101, 1:1000), p44/42 MAPK (Erk1/2) (9102, 1:1000), p-p38 MAPK (Thr180/Tyr182) (9211, 1:1000), p38 MAPK (9212, 1:1000), α-tubulin (2144, 1:2000), vimentin (3390, 1:1000), TACE (3976, 1:1000), Snail (3879, 1:1000), E-cadherin (3195, 1:1000), p-FAK (Tyr397) (3283, 1:1000), FAK (3285, 1:1000), p-STAT3 (Tyr705) (9131, 1:1000), STAT3 (9139, 1:1000), GAPDH (2118, 1:2000), β-actin (3700, 1:2000), p-DNA-PK (68716, 1:1000), DNA-PK (38168, 1:1000), PARP (9532, 1:1000), p-Histone H2A.X (Ser139) (9718, 1:1000), Histone H2A.X (2595, 1:1000), p-Chk2 (Thr68) (2197, 1:1000), Chk2 (2662, 1:1000), p53 (2524, 1:1000), caspase-7 (9492, 1:1000), caspase-3 (14220, 1:1000), AURK-A (14475, 1:1000); Santa Cruz Biotechnology (Dallas, TX): integrin β1/ITGB1 (sc-374429, 1:1000). Selumetinib (AZD6244, MEK1/2 inhibitor), osimertinib (AZD9291, EGFR^T790M), M3814 (DNA-PK inhibitor, DNA-PK-I) and ZM-447439 (AURK-A inhibitor, AURK-A-I), MK-4827 (PARP inhibitor, PARP-I), M4076 (ATM inhibitor, ATM-I), M6620 (ATM/ATR inhibitor, benzosertib) and M4344 (ATR inhibitor, ATR-I) were purchased from Selleck Chemicals (Selleckchem).

For cell cycle analysis and immunostaining, cells were collected and fixed in 70% ethanol followed by PBS washes; finally, they were dissolved in a hypotonic buffer containing propidium iodide. Samples were acquired on a Guava EasyCyte flow cytometer (Merck Millipore, Italy) and analyzed with a standard procedure using EasyCyte software. To detect the different proteins involved in DNA repair, we incubated fixed cells with the following antibodies: ATM (ab36810, ABCAM, UK); g-H2AX (2577, Cell Signaling, MA, USA); RAD51 (ab88572, ABCAM, UK); and DNA-PK (sc390698, SantaCruz Biotech, CA, USA). Cells were then incubated with corresponding secondary antibodies that were FITC conjugated. For each sample, 5,000 cells were evaluated on the Guava instrument.

For Western blotting (WB) experiments, whole protein extracts were prepared using RIPA buffer, as previously described [11 (link)]. Filters were hybridized with the following primary antibodies: ATM, BCL2, DNA-PK, MYC, p21 and PTEN by Santa Cruz Biotechnology (Dallas, TX, USA); NRF2 and GNL3 by Abcam, Cambridge, UK; γH2AX, phospho(p)-AKT, AKT, cleaved caspase-3 and cleaved PARP by Cell Signaling Technology, Danvers, MA, USA; BRD4 by Bethyl; CD133 by ThermoFisher Scientific, Waltham, MA, USA. Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Bethyl, Montgomery, TX, USA) were used. Protein signals were detected using WesternBright ECL kit (Advansta, Menlo Park, CA, USA), and visualized by ChemiDoc XRS+ (Bio-Rad). Tubulin (Santa Cruz Biotechnology) was used as a normalization control for equal loading of total proteins and cytoplasmatic fraction in SKOV3 cells. β-actin (Sigma) served as loading control for equal loading in UWB1.289 cells. Lamin B (Santa Cruz Biotechnology) was used as loading control for nuclear fraction. Densitometric analysis was performed using the Image Lab 5.1 software (Bio-Rad), as already reported [11 (link)].