Research Paper Volume 12, Issue 20 pp 20432—20444

Figure 2. Ku70 plus Ku80 double knockout potently inhibits LPS-induced production of pro-inflammatory cytokines in human macrophages. THP-1 human macrophages were transfected with lenti-CRISPR/Cas9-Ku70 KO construct plus lenti-CRISPR/Cas9-Ku80 KO construct, control cells were transfected with CRISPR/Cas9 control vector (“Cas9-C”), stable cells were established following puromycin selection, mRNA and protein expression of listed genes were tested by qPCR (A) and Western blotting (B); Cells were treated with LPS (500 ng/mL) or vehicle control (“C”) for indicated time, cell viability and death were tested by MTT (C) and Trypan blue staining (D), respectively; mRNA expression (E–G) and protein contents in the culture medium (H–J) of listed pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were tested by qRT-PCR and ELISA assays; Expression of listed proteins was quantified, normalized to the loading control (B). Data were expressed as mean ± standard deviation (SD, n=5). *p<0.05 vs. “C” treatment of “Cas9-C” cells. ^# p<0.05. LPS treatment of “Cas9-C” cells. Experiments in this figure were repeated three times, and similar results were obtained.