Fig 2: Ku70 and Ku80 silencing or KO inhibits LPS-induced pro-inflammatory cytokines production and NF?B activation in human PBMCs. The primary PBMCs were transfected with Ku70 shRNA lentivirus plus Ku80 shRNA lentivirus (“Ku70/Ku80-DKD”), or lenti-CRISPR/Cas9-Ku70 KO construct plus lenti-CRISPR/Cas9-Ku80 KO construct (“Ku70/Ku80-DKO”), expression of listed genes was shown (A and B); Cells were treated with LPS (500 ng/mL) or vehicle control (“C”) for indicated time, TNF-a mRNA expression and protein content in the culture medium were tested by qRT-PCR (C) and ELISA (D); Cell death was tested by Trypan blue staining assay (E); The relative NF?B activity was tested by p65 DNA-binding assay (I). THP-1 cells were treated with LPS (500 ng/mL) for 4h, nuclear lysate proteins were subjected to co-immunoprecipitation assay (“IP: Ku70/Ku80”) and Western blotting assay (“INPUT”) (F). Stable THP-1 human macrophages, bearing control shRNA lentivirus (“sh-c”), Ku70 shRNA lentivirus (“sh-Ku70”) and/or Ku80 shRNA lentivirus (“sh-Ku80”) (G), as well as the Ku70-expressing AAV plus the Ku80-expressing AAV (“Ku70/Ku80 D-OE”) or empty vector (“Vec”) (H), were treated with LPS (500 ng/mL) or vehicle control (“C”) for 6h, the relative NF?B activity was tested by p65 DNA-binding assay. Expression of listed proteins was quantified, normalized to the loading control (B). Data were expressed as mean ± standard deviation (SD, n=5). “Pare” stands for the parental control PBMCs (A–E, I). *p<0.05 vs. “C” treatment. # p<0.05. LPS treatment of control cells. Experiments in this figure were repeated four times, and similar results were obtained.

Fig 3: Ku70 plus Ku80 double overexpression enhances LPS-induced production of pro-inflammatory cytokines in human macrophages. mRNA and protein expression of listed genes in stable THP-1 human macrophages with the Ku70-expressing AAV plus the Ku80-expressing AAV (“Ku70/Ku80 D-OE”) or with control vector AAV (“Vec”) were shown (A and B); Cells were treated with LPS (500 ng/mL) or vehicle control (“C”) for indicated time, cell survival 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-a, IL-1ß and IL-6) were tested by qRT-PCR and ELISA; 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 “Vec” cells. # p<0.05. LPS treatment of “Vec” cells. Experiments in this figure were repeated four times, and similar results were obtained.

Fig 4: JMJD1A functions as a coactivator for c-Myc for the expression of DDR genes.a Knockdown of c-Myc in Rv1 cells reduced mRNA levels of indicated DDR genes. Cells were transduced with pLKO.1 control or c-Myc shRNAs for 48h. RNAs were analyzed by qRT-PCR for the indicated genes. b Knockdown of c-Myc in the JMJD1A-knockdown Rv1 cells did not further reduce the expression of DDR genes. Knockdown of JMJD1A alone or knockdown of both JMJD1A and c-Myc was performed on Rv1 cells for 48h. Cells were analyzed by qRT-PCR for the indicated DDR genes. c Coimmunoprecipitation between JMJD1A and c-Myc in Rv1 cells. Left panel: JMJD1A was immunoprecipitated from Rv1 cells, and analyzed by western blotting for the coprecipitation of c-Myc. Right panel: c-Myc was immunoprecipitated from Rv1 cells, and analyzed by western blotting for the coprecipitation of JMJD1A. d Coprecipitation of myc-JMJD1A with Flag-c-Myc but not with Flag-Max. myc-JMJD1A was coexpressed with Flag-c-Myc or Flag-Max in 293T cells. Cell lysates were subjected to immunoprecipitation (IP) with anti-Flag M2 beads. Bound proteins were analyzed by western blotting with myc or Flag antibodies. e Interaction of N-terminal transactivation domain of c-Myc with JMJD1A. myc-JMJD1A was coexpressed with the Flag-tagged truncation mutants of c-Myc and analyzed by Flag IP as described in (d). f Interaction of N-terminal or C-terminal half of JMJD1A with c-Myc. HA-tagged c-Myc was coexpressed with the Flag-tagged N-terminal half (N) or C-terminal half (C) of JMJD1A, and analyzed by the Flag IP. g Direct binding between purified c-Myc and JMJD1A. GST or GST-c-Myc bound on the glutathione agarose was incubated with the recombinant Flag-JMJD1A. The bound proteins were analyzed by western blotting with GST or Flag antibodies. h Enrichment of JMJD1A and c-Myc on the E box sites of indicated DDR genes in Rv1 cells. Cells were analyzed by ChIP using antibodies for control IgG, JMJD1A or c-Myc. The precipitated chromatin fragments were analyzed by qPCR with primers targeting the E box region of indicated DDR genes. Data were calculated as the percentage of input. i-k c-Myc knockdown in Rv1 cells reduced the enrichment of JMJD1A and increased the enrichment of H3K9me2 at the E box region of XRCC6 (i), PRKDC (j) or BARD1 (k) gene. l-n JMJD1A knockdown in Rv1 cells increased the enrichment of H3K9me2 and decreased the enrichment of c-Myc at the E box region of indicated DDR genes. *(p < 0.05), **(p < 0.01).

Fig 5: Reduced nuclear mobility of Topo IIß by ICRF-187. (A) Representative images of FRAP analysis of EGFP-Topo IIß. HeLa cells transiently expressing EGFP-Topo IIß or EGFP-Topo IIß carrying a G180I substitution were pretreated with either 20 µM ICRF-187 or DMSO. A small nuclear area indicated by a white circle in the nucleus was photobleached, and fluorescent images were obtained at the indicated time points. (B) Quantification of fluorescent signals of EGFP-Topo IIß in FRAP analysis. Fluorescent images were obtained at 5 sec intervals. Mean values with SD from seven individual experiments were plotted. (C) Tight association of endogenous Topo IIß with nuclear matrices in the presence of ICRF-187. HeLa cells pretreated with ICRF-187 were microirradiated with a pulsed UVA laser. The cells were incubated with detergent-containing (+CSK) or detergent-free (-CSK) buffer and subsequently subjected to fixation followed by coimmunofluorescence of Topo IIß and Ku70.