Fig 2: CEACAM1 is upregulated by NCS in an ATM-dependent manner. (a) Parental MCF-10A cells (MCF-10A) or MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA vector (CTRL) as a control were incubated for 4 h in the presence or the absence of 20 nM NCS. At the end of the incubation, total RNA was extracted and analyzed for the levels of CEACAM1 or ATM expression by quantitative real-time PCR. The values in the graph represent the mean±s.e.m. from three independent experiments. §P<0.005; #P<0.001. (b) MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA (CTRL) were incubated for 16 h in the presence or the absence of 5.46 nM NCS. At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or Nijmegen breakage syndrome 1 (NBS1) expression by western blotting. (c) Parental MCF-10A cells were incubated for 1 h in the presence of 10 µM KU-55933 or the same dilution of dimethyl sulfoxide (DMSO (solvent)) as a control, followed by 16 h incubation in the presence or the absence of NCS 5.46 nM. At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or NBS1 expression by western blotting. In b and c, numbers on the left indicate kDa. (d) MCF-10A cells were incubated in the presence of 5.46 nM NCS for the times indicated. At the end of the incubation, the cells were split into two parts and either processed for protein extraction or for total RNA purification. RNAs were analyzed for the levels of CEACAM1, CEACAM1 primary transcript (CEACAM1 int-ex), p21/Waf1 or BTG2 by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. Proteins were analyzed for the levels of CEACAM1, p53 Ser 15-p, p53 or ß-actin (lower panel). Numbers on the left indicate kDa. (e) MCF-10A cells were incubated for 1 h in the presence of 10 µM KU-55933 or the same volume of DMSO (solvent) as a control, followed by 4 h in the presence of the indicated concentrations of NCS. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates.

Fig 3: 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 PIAS2a. 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

Fig 4: Dominant negative effect of DSB repair by overexpression of zinc hook RAD50 Q672X mutant. RAD50 ubiquitously expressed in ESCC cell lines (a); knockdown of RAD50 in KYSE150 (b); overexpression of RAD50 mutants in KYSE150 and U2OS (c) induced higher expression of MRE11 in KYSE150 and MRE11/NBS1 in U2OS (d); delay of DSB repair ability indicated by ?H2AX foci formation in RAD50Q672X overexpression in U2OS after IR stimulation (e). *, **, *** indicate p < 0.05, 0.01, and 0.001, respectively. Endo: endogenous full-length RAD50, n.s.: nonspecific, Red asterisk: RAD50-Q672X.

Fig 5: Inhibition of Notch signaling rescues the migration and neurite defects of Nbs1-deficient neural cells. (A, B) Blockage of Notch signaling by inhibitors (10µM of DAPT or L685, 458) (A) or shRNA targeting Notch1 (B) improves migration deficiency of N2A cells caused by shNbs1 knockdown. The migratory activity was analyzed 24 h after drug treatment or shRNA transfection. Data are Mean ± SEM from four independent experiments. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Rescue of the neuronal migration in vivo. Brain cortical sections from P16 mice after IUE with indicated plasmids at E15.5 were imaged by confocal microscopy. The distribution of GFP+ cells in indicated regions of the cortex was quantified (right). The mean ± SEM of the percentage of GFP+ cells in indicated regions from three to five animals is shown (right panel). N: number of mice analyzed. Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis within marked corresponding layer group. *P < 0.05; ** or ##P < 0.01; *** or ### or $$$P < 0.001; n.s.: not significant. (D) Rescue of the neurite outgrowth in vivo. Brain cortical sections from P26 mice after IUE with indicated plasmids at E15.5 were imaged and GFP+ neurons in layers II/III of the cortex are presented (up penal). The mean ± SEM were quantified from indicated animals of each treatment. N: number of mice analyzed; n: number of neurons analyzed. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Inhibition of Notch signaling by DAPT ameliorates the arborization defects of Nbs1-iKO neurons. Primary neurons were isolated from two Nbs1+/f-CreERT2 (+/F) and two Nbs1f/f-CreERT2 (F/F) E15.5 embryonic brains and cultured with 4-OHT for four days. Cells were then treated with or without 10 µM of DAPT for another 4 days before analysis at 8dpo. The number of primary neurites (marked by MAP2) per neuron from 41~131 neurons (from at least two animals) are shown (right panel). The Mean ± SEM is shown. One-way ANOVA followed by Dunnett's multiple comparisons test was used for statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001. (F) Inhibition of Notch activity corrects neurite outgrowth defects of shNbs1 knockdown N2A. N2A cells were transfected with Tomato-tagged shRNA against luciferase (shLuc) or Nbs1 (shNbs1). Cells were induced to neuronal differentiation by 2.5 mM cAMP in the presence or absence of Notch inhibitors (10 µM of DAPT or L685, 458) 12 h after transfection. Cells were imaged 24 h after differentiation induction and represented imaged (left). Quantification of the total length of neurites and the areas of cell clusters was acquired by the IncuCyte3 system. Arrows mark the neurites. Data are presented as mean ± SEM of the relative neurite length (dividing total length by areas of cell cluster) from four experiments (right panel). Two-way ANOVA followed by Tukey's post-hoc test was used for statistical analysis. ***P < 0.001.