Fig. 6

Blocking SUMOylation sensitizes cancer cells to apoptosis induced by etoposide. a The cells shown in Fig. 6a were treated with the indicated doses of gamma radiation, and then the cells were grown for 14 days and subjected to an analysis of cell viability, as described in “Materials and methods.” The experiments were performed in triplicate. The results shown are averages of three independent experiments. Bars indicate the SEM. ***P < 0.001, Student’s t test. b U2OS cells with UBC9 stably knocked out were seeded in six-well plates and cultured for 24 h, treated with etoposide (20 µM) for 48 h, subjected to annexin V-FITC and propidium iodide staining and measured by flow cytometry (n = 3). Bars indicate the SEM. **P < 0.01, Student’s t test. c HCT116 cells seeded on six-well plates and cultured for 24 h were treated with 50 µM etoposide, 200 µM 2-D08, or both for 24 h, and then were analyzed as in b, n = 3. Bars indicate the SEM. **P < 0.01, Student’s t test. d HCT116 cells seeded on six-well plates for 24 h were treated with 50 µM etoposide, 10 µM ML-792, or both for 24 h, and then were analyzed as b, n = 3. Bars indicate the SEM. **P < 0.01, ***P < 0.001 Student’s t test. e–g HCT116 cells were subcutaneously injected into the flanks of nude mice to generate xenograft tumors (n = 6/group). After 8 days of the injection, the mice were treated intraperitoneally with 2-D08 (5 mg/kg) and/or etoposide (10 mg/kg) every 2 days for 10 days. Tumor volume was measured every 2 days (e). After 18 days, the tumors were removed from the nude mice and weighed. Bars indicate the SEM. *P < 0.05, **P < 0.01, Student’s t test. h Proposed model for the posttranslational regulation of hSSB1. In the case of DNA damage, hSSB1 is phosphorylated by ATM, acetylated by p300 and SUMOylated by PIAS2α. These three modifications prevent its ubiquitination and degradation by FBXL5. The SUMOylation of hSSB1 promotes the recruitment of NBS1 by hSSB1 to DNA damage sites to execute its functions in response to DNA damage. Both SIRT4 and HDAC10 are critical for the deacetylation of hSSB1, while SENP2 mediates the deSUMOylation of hSSB1