Znf830

U2OS-DRGFP cells, which carrying chromosomal integrated single-copy of I-SceI site, were used to detect protein recruitment to I-SceI endonuclease-induced DSB sites as described previously (24 (link)). Briefly, 24 h after transfection of indicated siRNA, cells were transfected with pCBASceI plasmids to introduce DSBs. Twenty four hours after pCBASceI transfection, ChIP assay was performed using Pierce Agarose ChIP Kit (Thermo Scientific, USA) and ZNF830 (HPA027211, Sigma) or CtIP (79809, Novus Biologicals) antibodies according to the manufacturer's instruction. The ZNF830 or CtIP associated DNA was detected by PCR using the primer at the I-SceI site. The primer sequence was: forward, 5′-TAC AGC TCC TGG GCA ACG TG-3′; reverse, 5′-TCC TGC TCC TGG GCT TCT CG-3′. The PCR program was: 95 °C for 5 min, 1 cycle; 95 °C for 45 s, 56 °C for 30 s and 72 °C for 30 s, 35 cycles; 72 °C for 10 min, 1 cycle. The amplified products were analyzed on a 1.2% agarose gel.

Human lung cancer tissue microarray (HLugA150cs02) was purchased from Shanghai outdo Biotech Co., Ltd (Shanghai, China). In the microarray, 75 lung cancer tissues and their adjacent lung tissues were conducted in formalin-fixed paraffin-embedded sections. Xenograft tumors were collected and fixed in formalin, embedded in paraffin and cut into 5 μm-thick sections. Sections were firstly deparaffinized with 100% xylene, followed by rehydration using gradient ethanol (100%, 95%, 70%, 30%, 0%). After inactivation of endogenous peroxidase and retrieval antigen, IHC staining was performed using R.T.U Vectastain Kit (Vector Laboratories) according to manufacturer's instructions. The primary antibodies were ZNF830 (1:100, Sigma), Ki-67 (1:800, Abcam) and γH2AX (1:500, EMD Millipore). The percentage of Ki-67 and γH2AX positive cells was determined from three separate fields in each of three independent tumor samples. The semi-quantitation of ZNF830 staining was carried out by immunoscore (27 (link)). The immunoscore was calculated by multiplying the intensity and percentage of positive staining. The intensity was defined as follows: 0, no appreciable staining; 1, weak intensity; 2, moderate intensity; 3, strong intensity; 4, very strong intensity. High ZNF830 was defined when immunoscore ≥ 100; Low ZNF830 was defined when immunoscore < 100.