Figure 6

DDR/DNA repair proteins show suboptimal activation in TICS compared with bulk cells. (a) Western blot analysis of H125 bulk cells and TICs, 1, 4 and 24 h after IR, and A549, H1299 and H23 2 h after IR with 8 Gy. Phosphorylated and total forms of DNA-PK, ATM and phosphorylated forms of the ATM substrates H2AX, KAP1 and Chk2 were analyzed. α-Tubulin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading controls. (b) Western blot analysis of H125 bulk cells and TICs, 4 h after bleomycin (2.5 μg/ml) or 24 h after cisplatin (1 or 10 μM) exposure. Phosphorylated and total forms of DNA-PK, ATM, phosphorylated forms of the ATM substrates H2AX, KAP1, SMC1, Chk2 as well as the levels of FANCD2 and Rad51 were analyzed. A representative blot of at least two independent experiments is shown. β-Tubulin or GAPDH was used as loading controls. (c) Western blot analysis of H125 and A549 bulk cells and TICs, 24 h after 8 Gy IR, or 1 or 10 μM continuous cisplatin exposure. FANCD2 and cdc25A was assayed. A representative blot of two independent experiments is shown. GAPDH was used as loading control. (d) Western blot analysis of PARP full-length and cleavage fragment 24 h after 10 μM of continuous cisplatin treatment, with or without 1 h pretreatment with DNA-PKcs (NU7026) or ATM (KU55933) inhibitors (both 10 μM). A representative blot of two independent experiments is shown. GAPDH was used as a loading control