Journal: Nucleic Acids Research

Article Title: RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling

doi: 10.1093/nar/gky1166

Figure Lengend Snippet: UV-induced NONO degradation is mediated by RNF8. ( A ) RNF8 depletion stabilizes NONO after UV damage. HeLa cells were transfected with control siRNA or RNF8 siRNA, and treated with UV (30 J/m 2 ) for 6 h. Cell lysates were processed for immunoblotting with the indicated antibodies. ( B ) UV irradiation increases the polyubiquitination of NONO. HeLa cells were irradiated with UV (30 J/m 2 ) for the indicated time points and processed for the in vivo ubiquitination assay (left panel). Immunoprecipitation experiment revealed that NONO interacted with SFPQ under the immunoprecipitation buffer condition containing 0.5% NP-40 (right panel). ( C ) UV-irradiation specifically ubiquitinates NONO but not SFPQ or PSPC1. In vivo ubiqutination assays of NONO, SFPQ and PSPC1 were performed with HeLa cells at the indicated time points following UV (30 J/m 2 ) irradiation. ( D ) Knockdown of RNF8 impairs in vivo NONO ubiquitination after UV DNA damage. HeLa cells transfected with control siRNA or RNF8 siRNA were treated with UV (30 J/m 2 ) for 4 h and processed for the in vivo ubiquitination assay. The ubiquitin-conjugated endogenous NONO was subsequently detected by Western blotting analysis using anti-Ub antibody.

Article Snippet: NONO siRNA duplexes (5′-CAGGCGAAGUCUUCAUUCA-3′) , RNF8 siRNA duplexes (5′-UGCGGAGUAUGAAUAUGAA-3′) ( , ), XPC siRNA duplexes (5′-GGA GGGCGAUGAAACGUUU-3′) ( ) and control siRNA duplexes (5′-CCUACGCCACCAAUUUGGU-3′) were synthesized at Bioneer. siRNA transfection was performed using Lipofectamine RNAimax reagent (Invitrogen) according to the manufacturer's instructions.

Techniques: Transfection, Control, Western Blot, Irradiation, In Vivo, Ubiquitin Proteomics, Immunoprecipitation, Knockdown

Journal: Nucleic Acids Research

Article Title: RNF8 mediates NONO degradation following UV-induced DNA damage to properly terminate ATR-CHK1 checkpoint signaling

doi: 10.1093/nar/gky1166

Figure Lengend Snippet: RNF8-mediated degradation of NONO is required for S phase progression by terminating ATR-CHK1 checkpoint signaling in UV-DDR. ( A ) RNF8 depletion causes sustained ATR-CHK1 checkpoint signaling during UV DNA damage. HeLa cells transfected with control siRNA or RNF8 siRNA were exposed to UV (30 J/m 2 ) radiation for the designated time points. Cells were lysed and processed for Western blotting analysis. ( B ) Ectopic expression of stable triple lysine mutant NONO leads to prolonged activation of ATR-CHK1 checkpoint signaling. HeLa cells were transfected with FLAG-NONO and FLAG-NONO K279/290/295R and then treated with UV (30 J/m 2 ) for the indicated time points. Cell lysates were probed with the indicated antibodies for Western blotting analysis. ( C ) Stabilization of NONO delays S-phase progression after UV DNA damage. HeLa cells transfected with FLAG-NONO and FLAG-NONO K279/290/295R were irradiated with UV (10 J/m 2 ) for the indicated time intervals, and DNA content was analyzed by FACS analysis using propidium iodide staining. The cell cycle profiles were quantified and depicted in the top right corner of the figures. ( D ) Clonogenic survival of U2OS-derived stable cell lines expressing the GFP-tagged NONO wild type or the triple lysine (3KR) NONO mutant after UV radiation. Curves show mean ± s.d. of three independent experiments. **** P < 0.0001; P -values represent two-way ANOVA results.

Article Snippet: NONO siRNA duplexes (5′-CAGGCGAAGUCUUCAUUCA-3′) , RNF8 siRNA duplexes (5′-UGCGGAGUAUGAAUAUGAA-3′) ( , ), XPC siRNA duplexes (5′-GGA GGGCGAUGAAACGUUU-3′) ( ) and control siRNA duplexes (5′-CCUACGCCACCAAUUUGGU-3′) were synthesized at Bioneer. siRNA transfection was performed using Lipofectamine RNAimax reagent (Invitrogen) according to the manufacturer's instructions.

Techniques: Transfection, Control, Western Blot, Expressing, Mutagenesis, Activation Assay, Irradiation, Staining, Derivative Assay, Stable Transfection

Journal: Nature communications

Article Title: Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair.

doi: 10.1038/ncomms16102

Figure Lengend Snippet: Figure 2 | HBO1 is required for GG-NER but not TC-NER. (a) Depletion of HBO1 caused defects of unscheduled DNA synthesis (UDS) in HeLa cells. Cells were irradiated with 15 J m 2 ultraviolet and incubated for 3 h with EdU. Ultraviolet damage-dependent EdU incorporation of G1 cells was quantified and is shown as % of shCtl cells. Cells (n ¼ 1,500) from nine independent experiments; error bars indicate means±s.d. **Po0.01 (Student’s t-test). (b) Depletion of HBO1 did not affect recovery of RNA synthesis (RRS) after ultraviolet irradiation. HeLa shCtl or shHBO1 cells (left upper) and normal or UVsS-A fibroblasts (left bottom) were transfected with indicated siRNA. RRS assay was performed as described in Methods. Relative RNA synthesis is shown as % of mock irradiated. Depletion of CSB and HBO1 from HeLa and UVsS-A and HBO1 from fibroblasts was confirmed by western blotting (right). Cells (n ¼ 300) from three independent experiments; error bars indicate means±s.d.

Article Snippet: Control, HBO1, DDB2 and XPC shRNA plasmids were purchased from Santa Cruz (sc-108065, sc-35530B-SH, sc-37799-SH and sc-37805SH).

Techniques: DNA Synthesis, Irradiation, Incubation, Transfection, Western Blot