Fig 2: In response to UV irradiation, SIRT6 interacts with DDB2. (A) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. ***P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo. HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies (B). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis (C). (D) SIRT6 interacts with DDB2 in vitro. Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 µl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. (E) A schematic representation of the SIRT6 fragments used in this study is shown. (F) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ?C-Flag (1-271 aa), SIRT6 ?N-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ?N?C-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. (G) A schematic representation of the DDB2 fragments used in this study is shown. (H) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. (I) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. **P < 0.01, n.s., not significant.

Fig 3: PSMD7 stabilizes RAD23B via deubiquitination in GC cells. (A) AGS cells were infected with lentiviruses carrying non-targeting (NT) shRNA or PSMD7 shRNA (shPSMD7-1 and shPSMD7-2) and analyzed by WB for RAD23B and XPC expression. (B) AGS cells were transfected with pcDNA3.1 vector carrying PSMD7 cDNA or an empty vector and analyzed by WB for RAD23B and XPC expression. (C) Co-IP assay confirmed the interaction between PSMD7 and RAD23B in AGS cells. (D) PSMD7 knockdown increased the ubiquitination of RAD23B in AGS cells. (E) AGS cells with or without PSMD7 silencing were treated with CHX (40 mg/mL) to block protein synthesis. WB was carried out to detect the RAD23B level 0, 1, 3, and 5 h after treatment. *P < 0.05.

Fig 4: hSSB2 depletion delays XPC recruitment, impairs RPA recruitment to UV damage sites and increases sensitivity to UVB exposure. (a) Representative images of HeLa cells probed for CPD (green, left panels) and RPA32 (red, middle panels) without UV exposure or 1 h and 3 h after UVC irradiation with a 5 µm filter. Cells were irradiated 48 h after negative control (si Control) and hSSB2 (si hSSB2) siRNAs transfection with UVC covered with 5 µm filter. DNA is stained with DAPI (blue, right panels). Average RPA32 fluorescent staining intensity at the CPD sites (± sem, n = 3) has been reported in graph (b). (c) Representative images of HeLa cells probed for CPD (green, left panels) and XPC (red, middle panels) without UV exposure or 15 min and 1 h after UVC irradiation. DNA is stained with DAPI (blue, right panels). Average XPC fluorescent staining intensity at the CPD sites (± sem) has been reported in graph (d). Scale bar represents 5 µm. (e,f) Measure of the average live cells (± sem, n = 3) 48 h after UVB exposure of HeLa cells transfected with negative control (si Control) or hSSB2 (si hSSB2) siRNAs (e) and U-2OS cells (f). Analysis was carried out with the Cytell imaging system 20 min after Hoechst and Propidium Iodide staining (****P-value < 0.0001 in a Student test).

Fig 5: Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. (A) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m2), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. (B) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 µM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. (C) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. (D) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. (E) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. (F) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m2) and harvested at the indicated time points, followed by western blot analysis.