Dna pkcs

NSCLC cell lines A549, HCC4006 and Calu-1 (three human lung adenocarcinoma cell lines), and SK-MES-1 and KLN205 (two human lung squamous carcinoma cell lines) were obtained from the Shanghai Institute for Biological Science (China). All cell lines were purchased between 2012 and 2015 and authenticated based on growth rate, morphology, and viability and were frequently confirmed to be mycoplasma free. The cells were cultured in RPMT 1640 media supplemented with 10% heat-inactivated fetal calf serum (FBS), L-glutamine and 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate. The cells were maintained in a humidified incubator in 5% CO₂ at 37 °C.
Antibodies to various antigens were as follows: CHK1, PCHK1 (S296), PCHK1 (S317), PCHK1 (S345), CHK2, PCHK2 (T68), PCHK2 (S516), Cdc25A, PCdc25C (S216), H2AX, γH2AX (S139), PH3 (S10histone), ATM, PATM (S1981), DNA-PKCs, PDNA-PKCs (S2056), and GAPDH were from Cell Signaling. FANCL, FANCD2, BRCA2, PARP1, RAD51, caspase-3, cleaved caspanse-3, PARP and cleaved PARP from Santa Cruz. Gemcitabine was from Hanson Pharmaceutic (China), Cisplatin from Yangtze River Pharmaceutic (China), MK-8776 from Selleck Chemicals, CHK2 inhibitor II from Abcam. Gemcitabine was dissolved in PBS. Cisplatin, MK-8776 and CHK2 inhibitor II were dissolved in DMSO and used at the specified concentrations.

Cells were cultured and treated with resveratrol for different time periods and different concentrations. Next, cells were lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 mg/mL PMSF, 2 mg/mL aprotinin, 2 mM leupeptin, and 1 mg/mL pepstatin) and protein concentration was determined using the Bradford assay. Protein extracts were resolved by SDS–PAGE (60 mg per lane) on a 10% polyacrylamide gel and transferred into immobilon membranes (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, membranes were incubated with 1:1000 dilutions of primary antibodies. We used antibodies against p-ATM, p-BRCA1 and γ-H2AX (Cell Signaling Technology, Inc., Danvers, MA, USA) as markers of DNA damage response; active-caspase 3 and cleaved PARP (Cell Signaling Technology, Inc., Danvers, MA, USA) to determine apoptosis marker; and finally, Rad50, Mre11, p-p95/NBS1, DNA-PKcs, and KU80 (Cell Signaling Technology, Inc, Danvers, MA, USA) as markers of DNA repair; Tubulin (Calbiochem, Darmstadt, Germany) was used as loading control.

Whole cell lysates were prepared using RIPA cell lysis buffer (9803, Cell Signaling) and 1 mM PMSF (8553s, Cell Signaling). Lin^- cells, cells were cultured for 24 h in StemSpan SFEM medium (09600, StemCell Technologies) containing 2% L-glutamine (25030-081GIBCO), 1% penicillin/streptomycin (15140–122, GIBCO), 100 ng/ml SCF (250-03-10UG, Peprotech) and 100 ng/ml TPO (315-14-10UG, Peprotech). Lin^- cells were exposed to TGFβ1 (5 ng/ml) or TGFβ3 (5 ng/ml) along with LSN3301240 (5 μM) during 2 h. Western blots were performed using the following antibodies SMAD2/3 (86855, Cell Signalling), phospho-SMAD2 (ab3849, Millipore), phospho-SMAD2/3 (8828s, Cell Signaling) and Vinculin (sc-25336, Santa Cruz) antibodies.
Bone marrow stromal cell lines were exposed to irradiation (5 or 10 Gy), MEK inhibitor PD0325901 (10 μM) or TGFβ inhibitor LSN3301240 (5 μM). Western blots were performed with the following antibodies DNA-PKcs, 53BP1, RAD51, phospho-SMAD3 (9502s, Cell Signaling Technology, Danvers, MA, USA), p-ERK and actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA). Antibodies for analysis of fetal liver were DNA-PKcs, 53BP1, pERK1/2, total ERK1/2, CD45 CD41 and Vinculin.