Histone H2A.X Antibody - #AF6187

FIGURE 8 Lysine demethylase 1A (KDM1A) cooperates with SUMOylated Ras-responsive element binding protein 1 (RREB1) to promote the expression of 5-fluorouracil (5-FU) targets and to activate Chk1-mediated DNA damage response (DDR). (A) Mass spectrometry identification of proteins interacting with RREB1 under 50 µM 5-FU treatment for 24 h or in normal condition (mock) in HCT116 cells with overexpressing Flag-RREB1. Co-immunoprecipitation (Co-IP) was conducted with anti-Flag antibody. (B, C) Validation of interaction in HCT116 (B) and HEK293T (C) cells in normal condition and 5-FU treatment. (D) ML-792 attenuated the interaction between RREB1 and KDM1A. (E) We generated Flag-tagged RREB1 truncated mutants to explore the fragment within RREB1 that is required for interaction with KDM1A. (F) We transfected pFlag-Control, pFlag-RREB1, or pFlag-RREB13KR into KDM1A-interfering colorectal cancer (CRC) cells. After 48 h, 5-FU was added to culture at a final concentration at 50 µM for further incubation for 24 h. Then total proteins were collected for western blot analysis. (G, H) Under 5-FU treatment, effect of RREB1 and RREB13KR on formation of γH2AX foci (G) or 53BP1 foci (H) were measured by immunofluorescence (IF) assay after KDM1A was interfered. siControl is a scramble sequence control. Scale bars: 10 µm. (I) Comet assay was applied to evaluate the effect of KDM1A silencing on RREB1-mediated DNA damage protection. Scale bars: 100 µm. (J) Colony formation assay under 1 µM 5-FU treatment for 10 days. *p < 0.05, **p < 0.01, ****p < 0.0001, ns, not significant.

Fig. 2.| Measure the effect of PolyG on silica-induced DNA damage and nucleolin expression in a mouse model of silicosis. (A) expression of nucleolin and DSBs marker γ-H2AX in mouse lung tissue as measured by Western blotting.

Fig. 3. Both electrophility and the ROS producing activity of emodin contributed to its toxicity in THLE-2 cells. (A) Effect of emodin on ROS generation in THLE-2 cells. Cells were treated with indicated concentra- tions of emodin for 24 h, and intracelluar ROS was monitored with DCFH-DA probe. Data were presented as means ± SD. *P < 0.05, **P < 0.01, compared with control. (B) Effect of catalase on emodin-induced cell toxicity in THLE-2 cells. Cells were treated with emodin in the presence or absence of catalase (17.5 IU/ mL) for 24 h. Cell viability was tested by MTT assay. Data were presented as means ± SD. **P < 0.01, ***P < 0.001, compared with the cor- responding control; ### P < 0.001, emodin and catalase cotreatment group vs. emodin treat- ment group. (C) Effect of emodin on protein thiol (protein-SH) levels in THLE-2 cells. Cells were treated with indicated concentrations of emodin for 24 h. Intracellular levels of protein- SH were measured. Data were presented as means ± SD. *P < 0.05, ***P < 0.001, compared with control. (D) Effect of emodin on the status of H 2 AX activation in THLE-2 cells. Cells were treated with emodin for 24 h, and equal amounts of total cell lysates (50 μ g) were loaded and subjected to immunoblot analysis. Data represent the means ± SD of three independent experiments. **P < 0.01, ***P < 0.001, compared with control group.

Figure 4 A combination of Chidamide and Bortezomib increases production of ROS dependent DNA damage and the changes of cell apoptosis and cycle pathway in MM cells. (A) ARP-1 cells were pretreated with or without NAC (15.0 mmol/L) for 2 hours at 37°C and then incubated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) for 24 hours, then ROS generation was detected. (B) ARP-1 cells were pretreated with or without 15 mmol/L NAC and then treated with Chidamide or Bortezomib alone or in combination, and cell viabilities were evaluated using CCK-8 assays. (C) The expression of γ-H2AX in ARP-1 cells treated with CHI (1.0 µmol/L) and/or BTZ (5.0 nmol/L) were determined by Western blot. (D) Representative images of γ-H2AX (Red) and nuclei (Blue) in ARP-1 cells treated with single agent or combination for 24 hours by immunofluorescence assay. Scale bars represent 20 µm. (E, F) Western blot analysis of the expressions of cleaved caspase3, cleaved caspase8, cleaved PARP-1 and HDAC1 in XG1 (E) and ARP-1 (F) cells after 48 hours treatment with single agent or in combination. Error bars indicate mean ± SD. **p < 0.01.