Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: BBR directly binds to and inhibits CSN5 activity. (A) The 293T or 293T expressing Flag-CSN5 cell lysates were incubated with BBR-biotin at 4 °C overnight, the lysates were used for streptavidin–agarose pull-down assays, and the precipitates were resolved by IB for CSN5. (B) The recombinant Flag–CSN5 proteins were incubated with BBR–biotin for 1 h at 37 °C, followed by IB with biotin (upper band) or Flag (lower band). (C) The recombinant Flag–CSN5 protein was incubated with BBR–biotin in the absence or presence of indicated concentration of unlabelled BBR for 1.5 h at 37 °C, and the mixtures were IB for biotin or flag. (D) SPR analysis of the binding between BBR and CSN5. Recombinant human CSN5 protein was immobilized on an activated CM5 sensor chip, BBR was then flowed across the chip. (E) CSN5 activity in an in vitro deubiquitination assay. The activity was measured by 7-amino-4-methylcoumarin (AMC) released from the fluorogenic substrate, ubiquitin–AMC ( n = 3). ∗ ∗ P < 0.01 and ∗ ∗ ∗ P < 0.001 compared with DMSO group. (F) IB analysis of the P53 and P27 levels in H460 cells treated with indicated concentration of BBR for 24 h. (G and H) H460 cells were transfected with siRNA control, siRNA targeting CSN5 for 24 h (G), or transfected with 2 μg empty vector, 2 μg Flag–CSN5 for 24 h (H), followed by BBR (10 μmol/L) treatment for 24 h, the PD-L1 expression level was determined by IB. Data shown are mean value of three independent experiments±standard error of mean (SEM).

Article Snippet: The plasmids pcDNA3-HA-Ub (18712) obtained from Addgene (Watertown, MA, USA), pCMV3-PD-L1-Myc (HG10084-CM) and pCMV3-CSN5-Flag (HG17128-CF) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Activity Assay, Expressing, Incubation, Recombinant, Concentration Assay, Binding Assay, In Vitro, Transfection, Control, Plasmid Preparation

Journal: Acta Pharmaceutica Sinica. B

Article Title: Berberine diminishes cancer cell PD-L1 expression and facilitates antitumor immunity via inhibiting the deubiquitination activity of CSN5

doi: 10.1016/j.apsb.2020.06.014

Figure Lengend Snippet: Glu76 of CSN5 is critical for binding to BBR. (A) Molecular docking model carried out by Discovery Studio 4.5 revealed that BBR binds to the JAMA domain of CSN5. (B) BBR forms an ionic bond with the backbone of Glu76 and interacts favorably with several residues including Met78, His138, Tyr143, and Asp151. (C) 293T-cells were transfected with WT-CSN5 and CSN5-mutant plasmids for 48 h, cell lysates were incubated with BBR–biotin at 37 °C for 2 h, followed by pull-down with streptavidin–agarose; the precipitates were then immunoblotted by flag antibody. (D) 293T-cells were co-transfected with Myc–PD-L1, HA–Ub and Flag–CSN5 WT or Flag–CSN5 E76A, followed by BBR treatment for 8 h. The ubiquitination of PD-L1 were analyzed by IB. (E) Proposed model of BBR-mediated PD-L1 degradation. BBR specific binds to and subsequently inactivates CSN5, which led to degradation of PD-L1 and activation of tumor-infiltrating T-cells.

Article Snippet: The plasmids pcDNA3-HA-Ub (18712) obtained from Addgene (Watertown, MA, USA), pCMV3-PD-L1-Myc (HG10084-CM) and pCMV3-CSN5-Flag (HG17128-CF) plasmids were purchased from Sino Biological Inc. (Beijing, China).

Techniques: Binding Assay, Transfection, Mutagenesis, Incubation, Activation Assay

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Focal amplification of COPS5 in TCGA breast cancer. Samples were sorted by COPS5 amplitude from the IGV of TCGA portal developed by the Broad Institute. ( b ) Correlation of mRNA level and copy number of COPS5 in TCGA breast cancer samples. ( c ) COPS5 copy number and ERα mRNA expression in TCGA breast cancer samples. ( d ) Immunohistochemistry of COPS5 in representative tamoxifen-untreated and refractory breast cancer. Anti-COPS5 antibody showed positive staining in the nucleus of tumour cells (scale bar, 50 μm). ( e ) χ 2 -calculation of the significance of COPS5-overexpression rates in tamoxifen-refractory and tamoxifen-untreated breast tumours. ( f ) Growth curve of parental and tamoxifen-resistant MCF7 clones established by in vitro dose-escalation 4OHT-treatment (refer to Methods) in the presence of 2 μM of 4OHT. Values are normalized to day 0 treatment. *Significant difference compared with other samples ( P <0.01 at both day 6 and day 8, Student's t -test). ( g ) Copy number of COPS5 genomic DNA normalized to LINE1 in parental and tamoxifen-resistant MCF7 clones. *Significant difference compared with parental MCF7 ( P <0.01, Student's t -test). ( h ) Expression of COPS5 mRNA in parental and tamoxifen-resistant MCF7 clones. Data were normalized to GAPDH and relative to COPS5 expression in the parental cell line which was set as 1. *Significant difference compared with parental MCF7 ( P <0.01, Student's t -test). ( i ) Western blot analysis and quantification of COPS5 protein expression in parental and tamoxifen-resistant MCF7 clones. ( j ) Quantitative RT–PCR analysis of ERα target genes pS2 and GREB1 in MCF7 clones after treatment with 500 nM 4OHT for 48 h. Data are presented as mean+s.e.m. of three biological replicates. *Significant difference compared with untreated parental MCF7 ( P <0.01, Student's t -test).

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Amplification, Expressing, Immunohistochemistry, Staining, Over Expression, Clone Assay, In Vitro, Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Western blot analysis of COPS5 expression in MCF7 cell lines stably engineered with empty vector (EV), WT or D151N COPS5 cDNA. ( b ) Quantitative RT–PCR analysis of ERα and its target genes pS2 and GREB1 by ERα ligand treatment (48 h) in MCF7 cell lines stably engineered with empty vector (EV), WT or D151N COPS5 cDNA. Data are presented as mean+s.e.m. of three biological replicates. E2: 1 nM, 4OHT: 500 nM. *Significant difference compared with basal (untreated) levels ( P <0.01, Student's t -test). ( c ) Growth of MCF7 cell lines stably engineered with empty vector (Par), WT or D151N COPS5 cDNA in the absence or presence of 4OHT (1 μM). Data are presented as mean+s.e.m. of three biological replicates. *Significant difference compared with other samples ( P <0.01, Student's t -test).

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Western Blot, Expressing, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Western blot analysis of proteins that were co-immunoprecipitated with anti-COPS5 antibodies in MCF7 parental cells. ( b ) Western blot analysis of NCoR and ERα expression in parental and tamoxifen-resistant MCF7 clones. P, parental cell line. ( c ) IHC of NCoR and COPS5 expression in representative tamoxifen-untreated and refractory breast cancer samples (scale bar, 50 μm). Anti-COPS5 and anti-NCoR antibodies showed nuclear and nuclear/cytoplasmic staining in tumor cells, respectively. ( d ) Quantification and statistical analysis of IHC staining of COPS5, NCoR and ERα in tamoxifen-untreated and refractory breast cancer samples.

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Western Blot, Immunoprecipitation, Expressing, Clone Assay, Staining, Immunohistochemistry

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Cycloheximide (CHX) chase assay in parental MCF7 cells transduced with vector, HA-COPS5-WT or HA-COPS5-D151N. Cells were treated with CHX and then analysed by western blotting. ( b ) Half-life of NCoR protein was calculated by quantification of the western blots of anti-NCoR and anti-actin (for normalization). *Significant difference compared with other samples ( P <0.05, Student's t -test). ( c ) Western blot analysis of COPS5, NCoR and ERα in MCF7 cells stably expressing empty vector (EV), WT or D151N COPS5 cDNA in the presence or absence of proteasome inhibitor MG132. ( d ) In vivo ubiquitination assay in HEK293T cells with overexpression of WT or D151N COPS5. Proteins pulled down by Ni-NTA beads and existed in whole cell lysates (WCL) are tested by western blot using antibodies shown on the left. His-Ubi, His 6 -tagged ubiquitin; Ubi-NCoR, polyubiquitinated NCoR protein. ( e ) Western blot analysis of NCoR expression on knockdown of COP9 subunits GPS1, CSN2 and CSN6 by specific shRNAs in 4OHT-resistant MCF7 clone #3. Ctl, control. ( f ) Quantitative RT–PCR of mRNA of ERα target gene pS2 in the MCF7 cell lines shown in e in response to 4OHT (500 nM) for 48 h. Data are presented as mean+s.e.m. of three biological replicates. All of the 4OHT-treated samples are significantly different from corresponding untreated samples ( P <0.01, Student's t -test).

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Transduction, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, In Vivo, Ubiquitin Proteomics, Over Expression, Knockdown, Control, Quantitative RT-PCR

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Western blot analysis of COPS5 and NCoR in MCF7 cells with transient expression of COPS5 and/or NCoR. ( b ) Quantitative RT–PCR analysis of ligand-regulated ERα target gene expression in parental and 4OHT-resistant MCF7 clones with expression of NCoR. *Significant difference compared with corresponding untreated basal levels ( P <0.01, Student's t -test). ( c ) Western blot analysis of COPS5, NCoR and ERα expression in the 4OHT-resistant MCF7 cells stably transduced with COPS5 shRNA or rescuing COPS5 cDNA. ( d ) Quantitative RT–PCR analysis of ERα target gene pS2 in the MCF7 cell lines shown in c in response to 4OHT (500 nM, 48 h). *Significant difference compared with the untreated CTL sample ( P <0.01, Student's t -test). ( e ) Western blot analysis of NCoR and ERα expression on knockdown by stable NCoR shRNAs in parental MCF7 cells. ( f ) Quantitative RT–PCR analysis of ERα target gene pS2 in the MCF7 cell lines shown in e in response to 4OHT (500 nM, 48 h). *Significant difference compared with the untreated CTL sample ( P <0.01, Student's t -test). Data are presented as mean+s.e.m. of three biological replicates. CTL, control.

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Targeted Gene Expression, Clone Assay, Stable Transfection, Transduction, shRNA, Knockdown, Control

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ChIP analysis of the pS2 promoter occupancy of ERα, COPS5, CSN2, NCoR and PCAF on stimulation with ERα ligands for 2 h in ( a ) parental MCF7 cells and ( b ) 4OHT-resistant MCF7 clone #3. E2, 1 nM; 4OHT, 1 μM. Data are presented as mean+s.e.m. from three independent experiments. *Significant difference compared with corresponding basal recruitment ( P <0.01, Student's t -test). ( c ) ChIP analysis of NCoR on stimulation with ERα ligands for 2 h in parental and engineered COPS5-overexpression MCF7 cells. *Significant difference compared with corresponding basal recruitment ( P <0.01, Student's t -test). ( d ) Kinetic ChIP analysis of ERα, COPS5 and NCoR on the pS2 promoter at 20-min interval in parental MCF7 or NCoR shRNA #1 cells. Data are presented as mean+s.d. from two independent experiments. ( e ) A schematic representation of the proposed molecular model of functional interactions between COPS5 and NCoR in ERα-dependent transcription based on the ChIP data (see text for details). TAM, tamoxifen.

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Over Expression, shRNA, Functional Assay

Journal: Nature Communications

Article Title: COPS5 amplification and overexpression confers tamoxifen-resistance in ERα-positive breast cancer by degradation of NCoR

doi: 10.1038/ncomms12044

Figure Lengend Snippet: ( a ) Western blot analysis of doxycycline (Dox)-inducible shRNA-mediated knockdown of COPS5 and complementary re-expression of COPS5 cDNAs in the cultured 4OHT-resistant MCF7 clone #1. ( b ) Colony formation assay of 4OHT-resistant MCF7 clone #1 on knockdown by COPS5 shRNAs and rescue by COPS5 cDNAs after 4OHT-treatment for 14 days at 1 μM. *Significant difference compared with basal growth ( P <0.01, Student's t -test). ( c ) Growth of 4OHT-resistant MCF7 clone #3 in response to COPS5 knockdown and 4OHT-treatment (1 μM). Data are presented as mean+s.e.m. from three independent experiments. *Significant difference compared with other samples ( P <0.01, Student's t -test). ( d ) Western blot analysis of the xenograft tumours derived from 4OHT-resistant MCF7 clone #3. Cells were stably engineered with doxycycline-inducible shRNAs. ( e ) Volumes of the MCF7 xenograft tumours on treatments as indicated. Data are presented as mean±s.d. of five mice each group. *Significant difference compared with other samples ( P <0.01, Student's t -test). Ctl, control; DOX, doxycycline; Indu-shRNA, inducible shRNA; Par, parental; TAM, tamoxifen.

Article Snippet: Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma.

Techniques: Western Blot, shRNA, Knockdown, Expressing, Cell Culture, Colony Assay, Derivative Assay, Stable Transfection, Control

Journal: Advanced Science

Article Title: HIC1 suppresses Tumor Progression and Enhances CD8 + T Cells Infiltration Through Promoting GSDMD‐induced Pyroptosis in Gastric Cancer

doi: 10.1002/advs.202412083

Figure Lengend Snippet: Jab1 specifically interacts with HIC1 and mediates the degradation of HIC1 in a ubiquitin‐proteasome manner at K517. (A) Whole‐cell extracts of MKN45 and HGC27 cells were isolated and subjected to a Co‐IP assay to investigate the interaction between endogenous Jab1 and HIC1. (B) IF assay was employed to detect the co‐localization of HIC1 and Jab1. (C) Western blot analysis revealed a significant reduction in HIC1 protein levels following transfection with Myc‐Jab1 overexpression plasmid. (D) Cell lysates were collected for electrophoresis following treatment with MG132 (20 µ m ) for 6 h. (E,F) Western blot was subjected to explore the impact of Jab1 overexpression on the protein stability of HIC1 for MKN45 and HGC27 cells, as compared to an empty vector. CHX (50 µ m ). (G) Myc‐Jab1, Flag‐HIC1, and HA‐ubiquitin plasmids were simultaneously transfected into cells and immunoprecipitated using HIC1 antibody prior to being immunoblotted. (H) Coomassie blue staining of the HIC1 protein complex with red arrows indicating the bands corresponding to HIC1 (76 kDa). (I) Ubiquitylation assays were carried out with FLAG‐tagged wild‐type HIC1 or lysine‐to‐arginine mutants K154R and K517R together with HA‐Ub plasmid. (J) Alignment of the region corresponding to amino acids 515–525 of human HIC1 across different species. (K) GC cells transfected with the indicated plasmids were subjected to a ubiquitination experiment with the indicated antibodies.

Article Snippet: For in vitro ubiquitination assay, recombinant human Jab1 (Proteintech, Ag25956), ubiquitin (ABclonal, RPO1960), UBE1(R&D Systems, E‐305‐025), UBE2B (YEASEN, 20428es10) and ATP (Solarbio, C0550) were utilized.

Techniques: Ubiquitin Proteomics, Isolation, Co-Immunoprecipitation Assay, Western Blot, Transfection, Over Expression, Plasmid Preparation, Electrophoresis, Immunoprecipitation, Staining

Journal: iScience

Article Title: Latent CSN-CRL complexes are crucial for curcumin-induced apoptosis and recruited during adipogenesis to lipid droplets via small GTPase RAB18

doi: 10.1016/j.isci.2023.106468

Figure Lengend Snippet: Key resources table

Article Snippet: CSN5(JAB1) , GeneTex , GTX70207.

Techniques: Recombinant, Plasmid Preparation, CRISPR, In Vitro, Mutagenesis, Software

Journal: Nature Communications

Article Title: Global and transcription-coupled repair of 8-oxoG is initiated by nucleotide excision repair proteins

doi: 10.1038/s41467-022-28642-9

Figure Lengend Snippet: a Representative time-lapse pictures of OGG1-GFP and GFP-DDB2 accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 50 μM Ro 19-8022 photosensitizer. b Quantification of accumulation kinetics of OGG1-GFP and GFP-DDB2 (as shown in a). c Schematic overview of the molecular interactions of DDB2 within the CUL4A-DDB1-RBX1 E3 ubiquitin ligase complex (CRL), which is required for the successive molecular interactions by ubiquitylation and subsequent DNA repair. The activation of CRL is mediated by covalent attachment of the ubiquitin-like activator NEDD8 on CUL4A and its proteolytic removal leads to the deactivation of ubiquitin ligase function. These crucial events can be fine-tuned by specific inhibitors MLN4924 (NAE1i) and SB-58-SN29 (CSN5i), acting on NEDD8-activating enzyme NAE1 and CSN5, respectively. d Representative time-lapse pictures of OGG1-GFP accumulation at micro-irradiated (405 nm laser) sub-nuclear area, indicated by arrows, in the presence of 10 μM Ro 19-8022 photosensitizer. Cells were pretreated with DMSO (CTR), NEDDylation inhibitor (NAE1i) or de-NEDDylation inhibitor (CSN5i) for 1.5 h. e Quantification of accumulation kinetics of OGG1-GFP (as shown in d). f Immunoblot analysis for DDB2, CUL4A, CSA and AQR (loading control) in MRC-5 expressing OGG1-GFP. Cells were treated with inhibitors as indicated in d. Scale bars: 5 µm. Data were normalized to the background and represent mean ± SEM from three independent experiments. Total number of cells “ n ” measured are indicated in figure legends. **** P < 0.001, analyzed by ROC curve analysis. Source data are provided as a Source Data file. (See also Supplementary Fig. ).

Article Snippet: The following inhibitors were used: NEDD8 neddylation activating enzyme inhibitor (NAE1 inhibitor, MLN4924, Boston Biochem) and CSN5-catalysed cullin de-NEDDylation inhibitor (CSN5 inhibitor, SB-58-SN29, kindly provided by Novartis) .

Techniques: Irradiation, Activation Assay, Western Blot, Expressing