Fig 2: The hSSB1 OB fold domain interacts with the p300 HAT domain. (A) HEK293T cells transfected with the indicated plasmids for 48 h were lysed with RIPA buffer. IP using anti-HA or anti-FLAG antibodies and western blotting with the indicated antibody were performed. (B) Schematic description of the p300 domains. (C) Schematic description of the hSSB1domains. (D) HEK293T cells cotransfected with Myc-hSSB1 and p300 mutants as indicated for 48 h were lysed with RIPA buffer and then analyzed by IP and western blotting. (E) HEK293T cells cotransfected with HA-p300 and hSSB1 1–113 or 114–211 mutants as indicated for 48 h were lysed with RIPA buffer and analyzed by IP and western blotting.

Fig 3: Inhibiting lysine 94 acetylation enhances cancer cell sensitivity to DNA damage agents. (A, B) Cells that express siRNA-resistant wild-type hSSB1 or its point mutant under the control of a tetracycline-inducible promoter were generated. The resulting cell lines were transfected twice with control or hSSB1 siRNAs. Following IR or CPT treatment, cells were permitted to grow for 14 days before staining. The experiments were performed in triplicate. The results shown are averages of three independent experiments. Bars indicate the SEM. **P < 0.01, ***P < 0.001, Student's t test. (C, D) Quantification of chromosomal aberrations in hSSB1-depleted HeLa cells expressing wild-type hSSB1 or hSSB1-K94R. The average of two experiments is shown. At least 50 cells were counted in each experiment. Bars indicate the SEM. **P < 0.01, Student's **t test. (E) U2OS cells were treated with either Eto (25 μM), C646 (10 μM) or combination for 24 h. The cells were then trypsinized and the percentage of apoptotic cells was detected by flow cytometry (n = 3), bars indicate the SEM. **P < 0.01, Student's t test. (F) U2OS cells were treated with C646 (10 μM) combined with Eto (10 μM), ADR (0.5 μM) or CPT (0.5 μM) for 48 h and the CCK8 activity of the cells was detected (n = 3), bars indicate the SEM. **P < 0.01, Student's t test. (G) Immunohistochemistry for hSSB1 and p300 was performed using 196 clinical colorectal cancer samples as described in the Materials and Methods section. Representative images of hSSB1 and of p300 expression levels in the same colorectal cancer sample. Case 1, both hSSB1 and p300 high expression. Case 2, both hSSB1 and p300 low expression. Scale bars: 100 μm. (H) A positive correlation was observed between hSSB1 and p300 in the 196 clinical colon cancer samples (P < 0.005, χ2 tests. R: Spearman correlation coefficient).

Fig 4: SIRT4 and HDAC10 deacetylate hSSB1. (A) HEK293T cells were transfected with FLAG-hSSB1 for 36 h, treated with TSA (2 μM) or NAM (5 mM) as indicated for 12 h, then lysed and analyzed by IP and western blotting. (B) hSSB1 was cotransfected with a series of FLAG-SIRTs into HEK293T cells for 48 h, then lysed and analyzed by HA-agarose IP and western blotting. (C) hSSB1 was cotransfected with a series of FLAG-HDACs into HEK293T cells for 48 h, then lysed and analyzed by HA-agarose IP and western blotting. (D) hSSB1 WT or K94R mutation plasmids were cotransfected with SIRT4 or HDAC10 for 48 h and acetylation levels were analyzed by western blotting.

Fig 5: Identification of the lysine 94 acetylation site through site mutation and mass spectrometry. (A) HEK293T cells were transfected with FLAG-hSSB1 mutated into arginine at the indicated lysine residue for 48 h and then analyzed by IP and western blotting. (B) FLAG-hSSB1 were overexpressed in HEK293T cells, purified by FLAG-agarose and isolated through SDS-PAGE. The hSSB1 band on the SDS-PAGE was analyzed by ESI-QUAD-TOF mass spectrometry. (C) hSSB1 WT or K94R mutant plasmids were cotransfected with p300 for 48 h and acetylation levels were analyzed by western blotting.