Total h2ax

For this study, SUV lamps (UVA-340nm) were purchased from Q-Lab Corporation (Cleveland, OH). The percentage of UVA and UVB of SUV lamps was measured by a UV meter and was 92.5% and 7.5% respectively. Commercial cefradine was obtained from Sigma-Aldrich (St Louis, MO, USA). The active TOPK human protein for the kinase assay was purchased from Millippore (Billerica, MA, USA). The pGEX-GST-H2AX human plasmid were purchased from Addgene Inc. The IL6 and TNF-α ELISA kit were purchased from Biosen Inc. (Beijing, China). Antibodies to detect total TOPK, total JNKs, total p38, total ERK1/2, total NF-κB p65, total H2AX, phosphorylated JNKs (Thr183/Thr185), phosphorylated p38 (Thr180/Thr182), phosphorylated ERK1/2 (Thr202/Tyr204), phosphorylated NF-κB p65 (S536) and phosphorylated H2AX (Ser139) were purchased from Cell Signaling Technology (Billerica, MA, USA). Antibodies to detect β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Colon 38 cells and HCT 116 (5×10⁵) were plated onto 6-well plates. After 24 hours, cells were treated as indicated in the figure legends. Cells were lysed in 2×SDS sample buffer (26.7mM pH 6.8 Tris-HCl, 1% SDS, 25% glycerol, 0.36M β-mercaptoethanol, and 0.05% bromphenol blue) and sonicated for 10 seconds to shear DNA. The whole-cell extracts were then electrophoresed through 15% or 8% SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Bio-rad). They were then incubated for 1 hour at RT with blocking solution (TBS, 0.1% Tween 20, and 3% nonfat milk), followed by a specific antibody to phospho-Histone H2A.X (Ser139) (1:2000, rabbit monoclonal mAb; Cell Signaling Technology, #9718), total H2A.X (1:2000, rabbit monoclonal mAb; Cell Signaling Technology, #7631), CASPASE 3 (D2R6Y) (1:1000, rabbit monoclonal mAb; Cell Signaling Technology, #14220), and LC3A (D50G8) (1:1000, rabbit monoclonal mAb; Cell Signaling Technology, #4599) overnight at 4°C. The membranes were then further incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000; Sigma) at RT for 2 hours, and signals were visualized by enhanced chemiluminescence (Perkin-Elmer Life Science). The phospho-Histone H2A.X and total H2A.X blots were reprobed with antibodies to β-Actin (1:2000; Sigma, A5316).