Ser139 p histone h2a x

KL and xenograft mice were perfused with 10 mM EDTA in PBS followed by 4% PFA. Both lung and xenograft tumors were extracted and fixed in 4% PFA for 12 hours and were followed by paraffin embedding. Tissue blocks were then sectioned (5 um) and subjected to heat-mediated antigen retrieval (citrate buffer, pH 6). Goat serum (Sigma) or donkey serum (Sigma) was used to block for 1 hour, and primary antibodies diluted were applied at 4°C overnight. Vectastain ABC (Vector Labs) with DAB substrate (Vector Labs) was used to optimize staining according to the manufacture’s protocol. The following primary antibodies were used: p63 (1:200; Biocare Medical; CM163A), p63 (1:100; R&D Systems AF-1916), GLUT1 (1:250; Alpha Diagnostic GT11-A), SGLT2 (1:1000; Abcam ab85626), TTF1 (1:1,000; Dako M3575), Ki67 (1:500; Cell Signaling Technology #12202), Cleaved Caspase-3 (1:200; Cell Signaling Technology #9664), Ser473-p-AKT (1:500; Cell signaling Technology #4058), Ser235/236-p-S6 (1:200; Cell Signaling Technology #4858) and Thr37/46-p-4EBP1 (1:200; Cell Signaling Technology #2855), Ser139-p-Histone H2A.X (1:1,000; Cell Signaling Technology #9718), 4-Hydroxynonenal (1:500; Abcam ab46545), CK5 (1:200; Abcam ab52635). Images were taken via Nikon Eclipse Ni-U microscope with NIS Elements imaging software (Nikon) and quantified using Fiji (NIH).

Heat-mediated antigen retrieval (citrate buffer, pH 6) was used for tissue sections from paraffin-embedded blocks. Samples were blocked with goat (Sigma) or donkey serum (Sigma) for 1 hour and diluted primary antibodies were applied overnight at 4^°C. Vectastain ABC (Vector Labs) with DAB substrate (Vector Labs) was used for staining according to the manufacturer’s instructions. The following primary antibodies were used: Ser139-p-Histone H2A.X (1:1,000; Cell Signaling Technology, 9718), CD31 (1:500, Cell Signaling Technology, 77699), β-Galactosidase (1:20000, abcam, ab9361), PDGFRα (Cell Signaling Technology, 3174), αSMA (1:500, Bio Care Medical, CM001A), and E-Cadherin (1:400, Cell Signaling Technology, 3195).