Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: The N-terminal acidic tail of CSN2 binds to the C-terminal basic canyons of cullins. (A) Pulldown of GST-CSN1/2/5/6 reveals coprecipitation of Cul1, -2, -3, and -4A with CSN2. (B) Direct interaction between E. coli-purified CSN2 and HEK293-purified GST-Cul4A. (C) Sequence the CSN2 N-terminal tail, the corresponding secondary structure was predicted by Psipred. Acidic residues in HsCSN2 are highlighted in red. (D) GST pulldown of CSN2 wild-type and its N-terminal deletion mutant Δ26. (E) The N-terminal tail of CSN2 is required for its ability to assemble CRL4A–CSN complexes. The shRNA targets the 3′-UTR of CSN2 and therefore does not influence transfected CSN2 (21). (F) Structure of Cul4A/Roc1 (PDB ID code 2HYE) shown in surface mode. The basic canyon is circled. (G) Mutations K465E and K675E abolish Cul4A binding to CSN2/5 but not to DDB1.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: Purification, Sequencing, Mutagenesis, shRNA, Transfection, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Inositol hexakisphosphate (IP6) generated by IP5K mediates cullin-COP9 signalosome interactions and CRL function

doi: 10.1073/pnas.1525580113

Figure Lengend Snippet: The N-terminal acidic tail of CSN2 binds to cullins. (A) Direct in vitro binding between CSN2 and GST-Cul4A wild-type, K705A, and K705R. (B) GST-Cul4A pulldown of recombinant CSN2 in the presence/absence of HA-Roc1. (C) High salt treatment markedly diminishes Cul4A binding to CSN subunits, with minimal effects on Cul4A–DDB1 interactions. (D) Sequence alignment of the CSN2 N-terminal tail, the corresponding secondary structure was predicted by Psipred. Acidic residues in HsCSN2 are highlighted in red. At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Dr, Danio rerio; Gg, Gallus gallus; Hs, Homo sapiens; Mm, Mus musculus. (E) Deletion of the N-terminal 17, but not the N-terminal 8, residues abolishes binding of CSN2 to cullins. (F) Pulldown of CSN2 wild-type and various mutants. (G) Amino acids 1–40 of CSN2 are sufficient to bind to Cul1, -2, -3, -4A. After transfection of the myc-cullin constructs, recombinant GST-rCSN21–40 on beads was incubated with cell lysates. (H) Recombinant CSN2 Δ26 is not pulled down by purified GST-Cul4A in vitro.

Article Snippet: The primary antibodies used were: Cul1, Cul2, HIF-1α, p21, p27 (Santa Cruz); Cul3 Cul4A, CSN5, K48-specific polyubiquitin, and DDB1 (Cell Signaling); GAPDH (Roche); IP5K and CSN2 (ProteinTech); NRF2 (R&D Systems); and GST (Sigma).

Techniques: In Vitro, Binding Assay, Recombinant, Sequencing, Transfection, Construct, Incubation, Purification

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 1. DDB1 interacts with and cleaved by NS3/4A. (A) DDB1 interacts with NS3/4A in overexpression system. The 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-Flag or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-Flag anti-HA. The lysates were analyzed by immunoblots with anti-DDB1 or anti-HA. (B) Endogenous DDB1 interacts with NS3/4A in JFH-1 infected cells. Huh-7 cells (5 107) were mock-infected or infected with JFH-1 (Multiplicity of Infection, MOI: 0.3) for 3 days. Coimmunoprecipitation was performed with anti-DDB1 or control IgG. The immunoprecipitates were analyzed by immunoblot with anti-DDB1 and anti-NS3. The lysates were analyzed by immunoblots with anti-DDB1 or anti-NS3.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Control, Western Blot, Infection

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 2. NS3/4A cleaves DDB1 at C378. (A) Cleavage of DDB1 by NS3/4A is inhibited by the NS3/4A inhibitor VX-950. The 293 cells were transfected with N-terminal or C-terminal Flag-tagged DDB1 (N-Flag-DDB1 or DDB1-C-Flag respectively) and HA-NS3/4A. The transfected cells were treated with VX-950 (0.2 mM) or left untreated for 1 day before immunoblot analysis with anti-Flag or anti-HA. (B) Alignment of the junction sequences of NS proteins of HCV and the potential NS3/4A cleavage sites in TC-PTP, VISA, TRIF and DDB1. (C) NS3/4A cleaves DDB1 at C378. The 293 cells were transfected with the indicated plasmids and cells lysates were analyzed by immunoblots with anti-Flag or anti-HA. (D) DDB1 N-terminal cleavage product migrated similarly to overexpressed DDB1(1–378) mutant. The 293 cells were transfected with the indicated plasmids, treated with VX-950 or left untreated for 1 day before immunoblot analysis with with anti-Flag or anti-HA.

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Transfection, Western Blot, Mutagenesis

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 3. DDB1 plays a critical role in HCV replication. (A) Overexpression of DDB1 potentiates HCV RNA replication. Huh-7 cells (1 106) were transfected with the indicated amounts of Flag-DDB1 plasmid for 24 h and then cells were split and mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-Flag or anti-b-actin. Graphs show mean7SD, n¼3. (B) Knockdown of DDB1 inhibits HCV RNA replication. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (C) Knockdown of DDB1 inhibits HCV protein expression. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 3 days, and the cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (D) Knockdown of DDB1 inhibits production of infectious HCV particles. Control or DDB1-RNAi knockdown Huh-7 cells were mock-infected or infected with JFH-1 for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show mean7SD, n¼3. (E) Knockdown of DDB1 inhibits RNA replication of HCV subgenomic replicon. Control or DDB1-RNAi knockdown Huh-7 cells and Huh-7 Con1 subgenomic replicon cells were cultured for 3 days. The cells (2 106) were collected and intracellular HCV RNA levels were determined by RT-qPCR and normalized to cellular GAPDH mRNA levels. Cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (F) DDB1 has no effects on HCV entry. Control or DDB1-RNAi knockdown Huh-7 cells were infected with HCVpp for 3 days (NC: Negative Control, HCVpp pakaging without HCV E1E2). The lysates of infected cells were assayed by luciferase reporter assays and immunoblots with anti-DDB1 or anti-b-actin. Graphs show mean7SD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Over Expression, Transfection, Plasmid Preparation, Infection, Quantitative RT-PCR, Western Blot, Knockdown, Control, Expressing, Staining, Cell Culture, Negative Control, Luciferase

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 4. DDB1 cleavage is required for HCV replication. (A) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) The indicated stable cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) The indicated stable cell lines were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh medium was added for 48 h. The JFH-1-containing medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D) Control or DDB1-RNAi knockdown Huh-7 cells were stably transduced with empty vector, DDB1, DDB1(C378R), off-target nonsense mutants of DDB1 or DDB1(C378R) respectively. Two days later, cells were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The cells lysates were also analyzed by immunoblots with anti-DDB1 or anti-actin. Graphs show meanþSD, n¼3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Stable Transfection, Infection, Quantitative RT-PCR, Western Blot, Staining, Control, Knockdown, Transduction, Plasmid Preparation

Journal: Virology

Article Title: DDB1 is a cellular substrate of NS3/4A protease and required for hepatitis C virus replication.

doi: 10.1016/j.virol.2012.10.025

Figure Lengend Snippet: Fig. 5. DDB1 cleavage products do not affect the HCV infection. (A) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. Intracellular HCV RNA levels were then determined by RT-qPCR and normalized to GAPDH mRNA levels. The uninfected cell lysates were analyzed by immunoblots with anti-DDB1 or anti-b-actin. Graphs show meanþSD, n¼3. (B) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 3 days. The cells were then analyzed by immunofluorescent staining with anti-E2 (red), and Hoechst (blue). (C) Huh-7 cells stably transduced with the indicated DDB1 truncation mutants were mock-infected or infected with JFH-1 (MOI: 0.3) for 24 h. The cells were completely washed and fresh complete medium was added for 48 h. The JFH-1 infected medium was collected and diluted for infection of Huh-7.5.1 cells. Three days later, cells were analyzed by immunofluorescent staining with anti-E2 and HCV titers were calculated by counting positive stained cells foci. Graphs show meanþSD, n¼3. (D–F) Control or DDB1-RNAi-#1 (targeted sequence is within the cDNA fragment encoding aa379–1140) transduced Huh-7 cells were further transfected with empty vector, Flag-DDB1(1–378), Flag-DDB1(379–1140*) (*, a RNAi off-target mutant),or a combination of Flag-DDB1(1–378) and Flag-DDB1(379–1140*) by Lipofectamine 2000. One day post transfection, the cells were split and mock infected or infected with JFH-1(MOI: 0.3) for 3 (D, E) or 1 (F) day. Intracellular HCV RNA levels (D), intracellular viral particles (E), or viral titers in the medium (F) were then determined as described in (A–C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Mouse monoclonal antibodies against Flag, HA, and b-actin (Sigma), HCV-NS3 (Abcam), HCV-Core(Santa Cruz Biotechnology); rabbit monoclonal antibodies against the C-terminus of DDB1 (Epitomics), rabbit polyclonal antibodies against the N-terminus of DDB1 (Santa Cruz Biotechnology, Proteintech Group); horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG and anti-rabbit IgG (Thermo); Alexa Fluor 555-conjugated anti-human IgG, Alexa Fluor 532-conjugated anti-mouse IgG; Hoechst 33258 (Invitrogen); and the NS3/4A inhibitor VX-950 (Selleck) were purchased from the indicated companies.

Techniques: Infection, Stable Transfection, Transduction, Quantitative RT-PCR, Western Blot, Staining, Control, Sequencing, Transfection, Plasmid Preparation, Mutagenesis

Journal: Nature Communications

Article Title: The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

doi: 10.1038/s41467-019-13808-9

Figure Lengend Snippet: a RepID-CRL4 localizes with BUB3 at the kinetochore in prometaphase cells. Immunofluorescence analysis of HCT116 RepID WT and KO cells was carried out in methanol-fixed cells using antibodies directed against CRL4 components (RepID, CUL4A, CUL4B, or DDB1; red signals) and BUB3 (green signal). CRL4 components do not associate with chromatin in the RepID KO cells because RepID, which recruits CRL4 to chromatin, is absent. Scale bar: 10 μm. b RepID-dependent CRL4 recruitment to chromatin throughout the cell cycle. Mitotic HCT116 RepID WT or KO cells were collected after a nocodazole block, reseeded in drug-free medium, harvested at the indicated time points, and chromatin-bound proteins were analyzed by immunoblotting and densitometry. Histone H3 and α-Tubulin were used as a loading control. c , d BUB3 is degraded in mitosis in RepID-expressing cells but not in RepID-deficient cells. HCT116 RepID WT or KO cells were released from mitotic block for the indicated time periods with or without the proteasome inhibitor MG132. Cell lysates were subjected to immunoblot analysis and densitometry. e BUB3 is ubiquitinated during mitosis in RepID-expressing cells but not in RepID-deficient cells. HIS-tagged ubiquitin plasmids were transiently transfected into HCT116 RepID WT and KO cells. Cells were collected at the indicated times after release from a nocodazole block, and his-tagged proteins from the released cells were isolated on Ni-NTA beads followed by immunoblot analysis. f Knockdown of CUL4 prevented the ubiquitination of BUB3 in RepID-expressing cells. HIS-ubiquitin plasmid and siRNA-CUL4 (directed against both CUL4A and CUL4B) were co-transfected into HCT116 RepID WT cells, which were synchronized, released, lysed, isolated with Ni-NTA beads, and analyzed for the presence of ubiquitinated BUB3 by immunoblotting as in e .

Article Snippet: Human FLAG-tagged expression plasmids for CUL4A (RC214798), CUL4B (RC206935), DDB1 (RC208372), BUB3 (RC200376), and RBBP7 (RC231256) were purchased from ORIGENE.

Techniques: Immunofluorescence, Blocking Assay, Western Blot, Control, Expressing, Ubiquitin Proteomics, Transfection, Isolation, Knockdown, Plasmid Preparation

Journal: Nature Communications

Article Title: The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

doi: 10.1038/s41467-019-13808-9

Figure Lengend Snippet: a RepID dissociates from CRL4 during mitotic progression. HCT116 cells were transfected with RepID FL expression plasmid (for constructs, see Fig. ). Cells were collected at the indicated times after release from a nocodazole block, and chromatin-bound fractions were extracted. Immunoprecipitation was performed using anti-FLAG antibody, and co-precipitated proteins were analyzed by immunoblotting. b RBBP7 is incorporated into CRL4 after mitosis. Chromatin fractions of FLAG-DDB1-transfected HCT116 cells released from mitotic block at the indicated time points were immunoprecipitated with anti-FLAG antibody, followed by immunoblot analysis. c RBBP7, but not other DCAFs, localizes to mitotic spindle during mitosis. Immunofluorescence analysis was performed using the indicated anti-DCAFs antibodies in HCT116 WT cells. Scale bar: 10 μm. d RBBP7 colocalizes with BUB3 during metaphase. HCT116 RepID WT or KO cells were subject to immunofluorescence analysis using anti-RBBP7, BUB3, and anti-CREST antibodies with/without treatment with a p97/VCP inhibitor (CB5083). Zoomed-in areas (marked by white squares) are shown at the bottom of the composite images (merged images of the zoomed-in area are shown to the left of the single-color zoomed-in images). Scale bar: 10 μm. Quantification of colocalization between RBBP7 and BUB3 by Pearson’s correlation coefficients (bottom panel) (* p value < 0.05, ** p < 0.01, *** p < 0.001, n.s. not significant, Student’s t test). e Depletion of RepID-CRL4 RBBP7 components or BUB3 overexpression can delay mitotic exit. Depleted or overexpressed HCT116 cells were released from a nocodazole arrest for 3 h and analyzed by flow cytometry. Percentages of cells in subG1, G1, G2/M, and >4 N phase are indicated. f RBBP7 facilitates the ubiquitination of BUB3 during mitosis. HIS-ubiquitin plasmids were transfected into HCT116 RepID WT cells together with siRNA-RBBP4 or si-RBBP7. Cells were synchronized, released, lysed, and proteins isolated on Ni-NTA beads as described in the legend for Fig. . Immunoblot analysis was carried out using anti-BUB3 antibodies. g A model describing the DCAF switch on chromatin during mitosis. RepID recruits CRL4 on chromatin before metaphase, bringing CRL4 in contact with BUB3. RepID then dissociates from CRL4, and RBBP7 is incorporated into CRL4 as a catalytic DCAF for BUB3 ubiquitination.

Article Snippet: Human FLAG-tagged expression plasmids for CUL4A (RC214798), CUL4B (RC206935), DDB1 (RC208372), BUB3 (RC200376), and RBBP7 (RC231256) were purchased from ORIGENE.

Techniques: Transfection, Expressing, Plasmid Preparation, Construct, Blocking Assay, Immunoprecipitation, Western Blot, Immunofluorescence, Over Expression, Flow Cytometry, Ubiquitin Proteomics, Isolation

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: DDB1 is associated with poor prognosis in colorectal cancer. ( A – D ) Analysis of the UALCAN database shows increased DDB1 expression in colorectal cancer (CRC). (A) DDB1 mRNA levels are significantly higher in primary colon adenocarcinoma (COAD) tissues compared to normal colon tissues. ( B ) DDB1 expression remains consistently elevated across various stages of CRC. ( C ) DDB1 protein levels are higher in CRC tissues. ( D ) Notably, DDB1 protein levels are exceptionally high in CRC samples with altered Wnt signaling. ( E ) Kaplan–Meier survival analysis reveals that high DDB1 expression is associated with poorer overall survival in CRC patients. ( F ) Gene Ontology (GO) analysis of DDB1-related genes reveals enrichment in pathways related to the cell cycle, DNA replication, and cell growth.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Expressing

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: DDB1 is required for CSC maintenance in colorectal cancer cells. ( A ) Western blot analysis of DDB1 expression in various CRC cell lines. HCT116 cells showed the highest expression. α-Tubulin served as a loading control. ( B ) qRT-PCR confirmed stable knockdown of DDB1 in HCT116 cells, with mRNA levels normalized to GAPDH. The expression level in control cells was set at 1 ( n = 3 independent experiments). ( C ) Relative mRNA expression of colorectal CSC markers (CD133, EPCAM, ALDH1A3, and ALDH1B1) in DDB1-knockdown (KD) and control cells. ( D ) Western blot showed decreased CD133 protein levels following DDB1 depletion. ( E ) Clonogenic proliferation was assessed in DDB1 -depleted HCT116 cells. Representative images of stained cells from plates with different well numbers are shown (upper panel). The proportion of viable cells was quantified by measuring colony-containing areas from the upper image (lower panel). Cell survival in the control was set at 100%. n = 3 independent experiments. ( F ) The tumor sphere formation assay revealed a decreased tumor-initiating capacity in DDB1 -depleted cells. Phase contrast images of tumorspheres derived from DDB1 -KD and control cells are shown in the upper panel. Quantification of sphere-forming cells in DDB1 -KD cells is shown (lower panel). The viability of control sphere cells was set at 100%. n = 5 independent experiments. Data are expressed as mean ± SEM of triplicate measurements; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Western Blot, Expressing, Control, Quantitative RT-PCR, Knockdown, Staining, Tube Formation Assay, Derivative Assay

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: DDB1 is O -GlcNAcylated by OGT in colorectal cancer cells. ( A - B ) Co-immunoprecipitation demonstrating interaction between DDB1 and OGT. HCT116 cells were co-transfected with 3xM-OGT and 3xF-DDB1, then treated with 100 nM Thiamet-G (OGA inhibitor; OGAi) for 24 h. Cell lysates were immunoprecipitated with anti-FLAG affinity gel and analyzed via Western blot using the specified antibodies. Symbols explained: 3xF, 3xFLAG-tagged empty vector; 3xM, 3xMYC-tagged empty vector; 3xF-SETD5, 3xFLAG-tagged-SETD5 plasmid; 3xM-OGT, 3xMYC-tagged OGT plasmid; αAlpha-tub, alpha-tubulin antibody. ( C – F ) DDB1 is O -GlcNAcylated by OGT. The O -GlcNAcylation of DDB1 was assessed through glycosylation assays using anti-Myc antibody ( C ) and sWGA beads ( D - F ). ( E ) The 100 nM OGAi was used to enhance O -GlcNAcylation of DDB1 before immunoprecipitation. Western blotting was conducted with the indicated antibodies. Symbols explained: αRL2, O -GlcNAc-specific antibody; sWGA, immunoprecipitation with succinylated wheat germ agglutinin beads to enrich O -GlcNAcylated proteins. ( F ) OGT depletion lowered the O -GlcNAcylation of DDB1. Cells transfected with siRNAs targeting luciferase (siCtrl) or OGT (siOGT) were introduced into HCT116 cells, along with either the 3xM vector or the 3xM-DDB1. After lysis with 1% NP40 buffer, proteins were immunoprecipitated with sWGA beads and analyzed by Western blot.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Glycoproteomics, Luciferase, Lysis

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: Ser-764 is the critical O -GlcNAcylation site on DDB1. ( A ) Predicted O -GlcNAcylation sites on DDB1 were identified using NetOGlyc and YinOYang tools. ( B ) glycosylation assays with domain-truncated DDB1 mutants showed that the BPC domain is the central region responsible for O -GlcNAcylation. The schematic diagram (upper panel) displays wild-type DDB1 (WT) and its three truncated mutants: 1–392 (BPA domain), 1–708 (BPA–BPB domains), and 1–1043 (BPA–BPB–BPC domains). ( C - D ) Site-directed mutagenesis was used to individually replace four candidate residues (T758, S762, S763, and S764) with alanine. Flag-tagged WT-DDB1 or glycosylation-defective DDB1 mutants were transfected into 293T cells and treated with 100 nM of OGAi for 24 h. Glycosylation assays using either FLAG-affinity gel ( C ) or sWGA beads ( D ) showed that O -GlcNAcylation was significantly reduced in the S764A mutant compared to WT and other point mutants, confirming Ser-764 as the key glycosylation site on DDB1.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Glycoproteomics, Mutagenesis, Transfection

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: O -GlcNAcylation of DDB1 boosts its interaction with CUL4A and p53 but not with CUL4B. ( A ) 293T cells were co-transfected with 3xF-CUL4A and 3xM-DDB1, then treated with either 50 nM OGT inhibitor (OSMI-1; OGTi) or 100 nM OGA inhibitor (Thiamet-G; OGAi) for 24 h to modulate DDB1 glycosylation. Increased glycosylation via OGAi enhanced the interaction between DDB1 and CUL4A, while decreased glycosylation via OGTi reduced it. ( B ) Under the same glycosylation-modifying conditions, the interaction between DDB1 and CUL4B remained unchanged, indicating that O -GlcNAcylation does not influence DDB1–CUL4B binding. ( C ) Immunoprecipitation analysis showed that the glycosylation-deficient mutant of DDB1 has significantly less binding to CUL4A compared to wild-type DDB1. 3xF-DDB1 (WT or the mutant) and 3xM-CUL4A were co-expressed in 293T cells and treated with 100 nM OGAi for 24 h. ( D ) The interaction between DDB1 and p53 was increased by OGAi-induced glycosylation in cells expressing wild-type DDB1 but not in cells with the mutant. 3xF-DDB1 (WT or the mutant) and 3xM-p53 were co-transfected into 293T cells, followed by 100 nM OGAi treatment for 24 h. Lysates were immunoprecipitated with an anti-p53 antibody and analyzed by Western blot using the indicated antibodies.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Transfection, Glycoproteomics, Binding Assay, Immunoprecipitation, Mutagenesis, Expressing, Western Blot

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: O -GlcNAcylation of DDB1 promotes p53 degradation. ( A ) Western blot analysis showing increased p53 protein levels in HCT116 cells stably expressing DDB1 shRNA compared to control. ( B ) Cycloheximide (CHX) chase assay demonstrating increased stability of p53 protein in DDB1 -depleted HCT116 cells. Cells were treated with 50 µg/mL CHX and harvested at the indicated time points. ( C ) Western blot analysis of p53 levels in HCT116 cells transfected with either FLAG vector control (3xF-Vec) or FLAG-DDB1 (3xF-DDB1), followed by treatment with OGT inhibitor (OGTi, 50 µM) or OGA inhibitor (OGAi, 100 nM) for 24 h. OGT inhibition resulted in increased p53 accumulation, while OGAi had no significant effect. ( D ) HCT116 cells were co-transfected with siRNA targeting luciferase (siLuc) or OGT (siOGT), along with either 3xF-Vec or 3xF-DDB1. Western blotting showed elevated p53 levels upon OGT depletion, confirming that OGT-mediated glycosylation of DDB1 regulates p53 stability. ( E ) Polyubiquitination of p53 is promoted by wild-type DDB1 but not by the glycosylation-deficient mutant (S764A). HCT116 cells were co-transfected with HA-ubiquitin (HA-Ub), 3xM-p53, and either wild-type DDB1 or the S764A mutant. Cells were treated with 100 nM OGAi for 24 h and 10 µM MG132 (proteasome inhibitor) for 6 h before harvest. Cell lysates were immunoprecipitated with an anti-p53 antibody and then immunoblotted with an anti-HA antibody to detect ubiquitinated p53.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Control, Transfection, Plasmid Preparation, Inhibition, Luciferase, Glycoproteomics, Mutagenesis, Ubiquitin Proteomics, Immunoprecipitation

Journal: Scientific Reports

Article Title: O- GlcNAcylation of DDB1 at Ser-764 by OGT promotes cancer cell stemness in colorectal cancer through increased polyubiquitination of p53

doi: 10.1038/s41598-025-30772-1

Figure Lengend Snippet: DDB1 influences CSC phenotypes via p53 regulation. ( A ) qRT-PCR analysis of CD133 mRNA expression in HCT116 cells stably expressing DDB1 shRNA, transfected with either siRNA targeting p53 (sip53) or a 3xM-p53 overexpression construct. DDB1 knockdown decreased CD133 expression, which was rescued by p53 knockdown and further suppressed by p53 overexpression. CD133 mRNA levels were normalized to GAPDH and are presented relative to the control (set as 1). n = 3 independent experiments. ( B ) Western blot analysis of CD133 protein levels under the same conditions. α-Tubulin was used as a loading control. ( C ) Tumorsphere formation assay showing that p53 knockdown rescues the decreased sphere-forming capacity caused by DDB1 depletion (left panel), whereas p53 overexpression further impairs tumorsphere formation (right panel). Sphere viability in control cells was set to 100%. n = 3 independent experiments. ( D ) Clonogenic assay showing that colony formation suppressed by DDB1 depletion is restored by p53 knockdown (upper panel), but not by p53 overexpression (bottom panel). Colony viability in control cells was set to 100%. n = 3 independent experiments. Data are shown as mean ± SEM of triplicate measurements; * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For DDB1-knockdown HCT116 cells, we cloned a pair of DDB1 shRNA targeting the coding sequence of DDB1 into a lentivirus-based pLKO.1 TRC cloning vector (Addgene; kindly provided by Dr. David Root) and produced lentiviral particles in HEK293FT cells to create stable knockdown cell lines.

Techniques: Quantitative RT-PCR, Expressing, Stable Transfection, shRNA, Transfection, Over Expression, Construct, Knockdown, Control, Western Blot, Tube Formation Assay, Clonogenic Assay

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: DDB1 and CRBN enhance ClC-2 protein degradation. ( A , B ) Co-immunoprecipitation of Myc-ClC-2 and Flag-DDB1 ( A ), as well as of Flag-ClC-2 and HA-tagged CRBN (HA-CRBN) ( B ), in HEK293T cells. Flag-DDB1 and HA-CRBN were recognized with α-Flag and the anti-HA antibody (α-HA), respectively. ( C ) Interaction of endogenous CRBN with ClC-2 in the rat brain. Whole brain lysates were immunoprecipitated with the anti-ClC-2 antibody (α-ClC-2), followed by immunoblotting of the immunoprecipitates with α-ClC-2 or the anti-CRBN antibody (α-CRBN). Co-immunoprecipitation of CRBN was achieved by using α-ClC-2 but not by rabbit IgG (α-IgG). l.c.: IgG light chain. ( D – E ) (Upper panels ) Representative immunoblots showing the effect of Flag-DDB1 ( D ) or HA-CRBN ( E ) co-expression on the Myc-ClC-2 protein level. Co-expression with the Flag or the HA vector was used as the control experiment. ( Lower panels ) Quantification of the relative ClC-2 protein levels. Values from the DDB1 or the CRBN co-expression groups ( hatched bars ) were normalized to those for the corresponding vector control ( clear bars ) (*, t test: p < 0.05; n = 7–10). ( F ) ( Upper panel ) Representative immunoblots showing the lack of an effect of Flag-DDB2 co-expression on the Myc-ClC-2 protein level. Co-expression with the Flag vector was used as the control experiment. ( Lower panel ) Quantification of relative ClC-2 protein levels ( t test: p > 0.05; n = 3). ( G ) Representative immunoblots showing the effect of shRNA knockdown of endogenous DDB1 (shDDB1) or CRBN (shCRBN) in HEK293T cells on Myc-ClC-2. Infection with shLacZ was used as the control experiment. The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Infection

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: DDB1 and CRBN reduce ClC-2 protein stability. Representative immunoblots showing the effect of vector ( A ), DDB1 ( B ), or CRBN ( C ) co-expression on ClC-2 protein turnover kinetics in HEK293T cells. Transfected cells were subject to different treatment durations (0 to 8 h) of the protein synthesis inhibitor cycloheximide (CHX). Protein densities were normalized to those of corresponding no-treatment controls at 0 h. The numbers on the immunoblot denote relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, Plasmid Preparation, Expressing, Transfection

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Endogenous expression of CUL4 E3 ligase in testes and Leydig cells. Representative immunoblots showing the endogenous expression of ClC-2 ( A ), CUL4A ( B ), CUL4B ( C ), DDB1 ( D ), and CRBN ( E ) in lysates prepared from mouse testes or cultured mouse Leydig cells. Approximately 30 µg of protein was loaded into each lane.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Expressing, Western Blot, Cell Culture

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: CUL4 and DDB1 promote the degradation of endogenous ClC-2. Representative immunoblots showing the effect of shRNA knockdown of CUL4A (shCUL4A) ( A ), CUL4B (shCUL4B) ( B ), or DDB1 (shCDDB1) ( C ) on endogenous ClC-2 expression in MA-10 cells. Infection with shLacZ was used as the control experiment. The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, shRNA, Knockdown, Expressing, Infection, Control

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Aldosteronism-associated gain-of-function alteration in ClC-2 proteostasis. ( A ) ( Upper panel ) Representative immunoblot comparing the protein expression of Myc-ClC-2 WT, aldosteronism-related G32D mutant, and leukodystrophy-related G511R mutant overexpressed in HEK293T cells. ( Lower panel ) Quantification of relative ClC-2 protein levels. Protein density was standardized as the ratio of the ClC-2 signal to the cognate tubulin signal. Values from the mutant groups ( hatched bars ) were then normalized to those for the corresponding WT control ( clear bars ). Asterisks denote a significant difference from the WT (*, t test: p < 0.05; n = 7–9). ( B ) ( Upper panel ) Representative immunoblot showing the effect of the 24-h treatment of 10 μM lenalidomide on the Myc-ClC-2 G32D mutant. ( Lower panel ) Quantification of the relative ClC-2 G32D protein levels (*, t test: p < 0.05; n = 7). ( C ) ( Upper panel ) Representative immunoblot showing the effect of HA-CRBN or Flag-DDB1 co-expression on the Myc-ClC-2 G32D mutant. ( Lower panel ) Quantification of the relative ClC-2 G32D protein levels (*, t test: p < 0.05; n = 4–6). ( D ) Representative immunoblot showing the enhanced surface expression of the Myc-ClC-2 G32D mutant, as well as its reduction by CRBN co-expression. Cell lysates from surface-biotinylated intact HEK293T cells were subject to either direct immunoblotting analyses ( Total ) or streptavidin pull-down prior to immunoblotting ( Surface ). The numbers on the immunoblot denote the relative ClC-2 protein levels.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Western Blot, Expressing, Mutagenesis, Control

Journal: Cells

Article Title: CUL4-DDB1-CRBN E3 Ubiquitin Ligase Regulates Proteostasis of ClC-2 Chloride Channels: Implication for Aldosteronism and Leukodystrophy

doi: 10.3390/cells9061332

Figure Lengend Snippet: Schematic model of endoplasmic reticulum (ER)-associated degradation of ClC-2. In this schematic diagram of the regulation of ClC-2 proteostasis by ER quality control, the scaffold protein CUL4A/B forms a protein complex with the adaptor protein DDB1 and the substrate receptor protein CRBN. CUL4A/B also interacts with the RING-finger protein ROC, which in turn recruits the E2 ubiquitin conjugating enzyme (E2) that transfers ubiquitin (Ub) for covalent linkage to a substrate protein. We propose that, through the direct interaction between CRBN and ClC-2, the CUL4A/B-DDB1-CRBN E3 ubiquitin ligase complex catalyzes the ubiquitination of misfolded ClC-2 proteins. Ubiquitinated ClC-2 is subsequently targeted for proteasomal degradation. Loss-of-function, leukodystrophy-causing mutations may instigate substantial protein misfolding, leading to enhanced degradation of mutant ClC-2 proteins. In contrast, gain-of-function aldosteronism-causing mutations appear to facilitate protein stability, thereby reducing proteasomal degradation of mutant ClC-2 channels. The CRBN-targeting immunomodulatory drug lenalidomide effectively promotes, whereas the cullin E3 ligase inhibitor MLN4924 significantly attenuates, proteasomal degradation of both ClC-2 WT and disease-associated mutant proteins.

Article Snippet: Myc-tagged ClC-2 in the pcDNA3 vector was generated by inserting the epitope sequence between the residues V420 and E421 in the extracellular linker between helices L and M. Other cDNA constructs employed in this study include pcDNA3.1-Flag dominant-negative human cullin 1/2/3/4A/4B/5 (Addgene 15,818–15,823, Watertown, MA, USA), pcDNA3-Myc human cullin 4A/4B (Addgene 19,951, 19,922, Watertown, MA, USA), pcDNA3-HA lysine-less human ubiquitin (kindly provided by Dr. Chihiro Sasakawa, University of Tokyo, Tokyo, Japan), pcDNA3-Flag human DDB1 (Addgene 19,918, Watertown, MA, USA), pcDNA3-Flag human DDB2 (kindly provided by Dr. Show-Li Chen, National Taiwan University, Taipei, Taiwan), and pcDNA3-HA rat cereblon (kindly provided by Dr. Chul-Seung Park, Gwangju Institute of Science and Technology, Gwangju, Korea).

Techniques: Control, Ubiquitin Proteomics, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: A clinically compatible drug‐screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

doi: 10.15252/emmm.202114552

Figure Lengend Snippet: Schema of experimental design. Organotypic cultures with established brain metastases from H2030‐BrM cells were treated with DEBIO‐0932 at 1 µM for 6 h and subjected to laser capture microdissection (LCM) and proteomic profiling. Representative image of a fully established brain metastasis from H2030‐BrM before and after laser capture microdissection (LCM). The dotted line delimits the metastasis. Scale bar: 100 µm. Volcano plot with deregulated proteins (red: upregulated; green: downregulated) found in brain metastases treated with DEBIO‐0932 compared to DMSO ( n = 3 biological replicates (mice) per condition, n ≥ 12 brain metastases per mouse were pooled together). Proteins with a P < 0.05 and a log 2 ratio > 1 or < −1 were defined as deregulated. Gray dotted lines indicate P value and log 2 ratio cut offs. The names of the top deregulated proteins are shown. GSEA of top 25 upregulated (red) and downregulated (green; only four fulfill the filter) pathways upon DEBIO‐0932 treatment. Those biological processes represented with more than one signature are labeled with colored lines. Representative images showing AHR and DDA1 levels in brain metastases (generated by intracardiac inoculation of H2030‐BrM) found at endpoint of vehicle and DEBIO‐0932‐treated animals. This result was reproduced in 2 independent staining with different brains. BB: bisbenzamide. Scale bars: low magnification (HSP90 and GFP), 50 µm; high magnification (DDA1), 6 µm (dotted lines). Representative images of squash preparations showing nuclear AHR and UBE4B in established brain metastases from H2030‐BrM generated by intracardiac inoculation. Scale bar: 5 µm. The dashed line surrounds the nucleus. Kaplan–Meier curves showing significant correlation between worse survival post‐brain metastasis and high expression levels of the HSP90 signature ( AHR , DDA1 , UBE4B , GPATCH8 ) in a cohort of 45 breast cancer brain metastasis patients. Representative images (selected cases obtained from Fig EV6M) and histological score of AHR, DDA1 and UBE4B in human brain metastases ( n = 16) according to the signal intensity of the corresponding protein in cancer cells. Schema of the experimental design. H2030‐BrM cells carrying the corresponding shRNA against AHR or the non‐targeting control were inoculated intracardially into nude mice. Ex vivo BLI of brains and thoracic regions were analyzed 5 weeks after injection of cancer cells. Brains were processed for histological analysis. Representative images of brains and thorax from shControl and sh AHR #1 mice at the endpoint of the experiment. Quantification of ex vivo BLI of brains and thoracic regions at the endpoint of the experiment. Values are shown in box‐and‐whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 9 shControl mice and n = 10 sh AHR #1 mice). P value was calculated using two‐tailed t ‐test. Quantification of the bioluminescence signal emitted by H2030‐BrM established metastases in organotypic cultures at Day 7 normalized by the initial value obtained at Day 0 and normalized to the organotypic cultures treated with DMSO. Day 0 is considered right before addition of the treatment or DMSO. Values are shown in box‐and‐whisker plots where each dot is an organotypic culture and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 17 organotypic cultures treated with DMSO; n = 18 organotypic cultures treated with BAY‐218, 2 independent experiments). P value was calculated using two‐tailed t ‐test. Schema depicting the evaluation of a clinical cohort composed of 251 ER + breast cancer primary tumors with follow‐up to determine the correlation of UBE4B, DDA1 or AHR with relapse. Representative images of primary tumors with high (red dot) or low (gray dot) UBE4B levels. A few cases of matched primary metastases allowed to evaluate the HSP90‐dependent protein. Scale bar: 100 µm. H‐score analysis of UBE4B in primary tumors with (red) or without (gray) associated relapse. Values are shown in a scattered plot where each dot is a primary tumor and the line corresponds to the median ( n = 100 primary tumors with relapse; n = 147 primary tumors without relapse). P value was calculated using two‐tailed t ‐test. Kaplan–Meier curve comparing relapse‐free survival of primary tumors with high and low values of UBE4B. P value was calculated using log‐rank (Mantel‐Cox) test.

Article Snippet: Slides were incubated with the corresponding primary antibodies as follows: HSP90α/β (clone F‐8, 1:3,000; sc‐13119, Santa Cruz Biotechnology, 8 min), DDA1 (1:2,400; 14995‐1‐AP, ProteinTech), UBE4B (1:500; ab97697, Abcam) and AHR (1:400; 031714, USBiologicals Life Sciences).

Techniques: Laser Capture Microdissection, Labeling, Generated, Staining, Expressing, shRNA, Control, Ex Vivo, Injection, Whisker Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: A clinically compatible drug‐screening platform based on organotypic cultures identifies vulnerabilities to prevent and treat brain metastasis

doi: 10.15252/emmm.202114552

Figure Lengend Snippet: A Representative images showing RPLP1 levels in brain metastases (generated by intracardiac inoculation of H2030‐BrM) found at endpoint of vehicle and DEBIO‐0932‐treated animals. This result was reproduced in two independent staining with different brains. Scale bars: 50 µm. B Quantification of RPLP1 and AHR levels shown in (Figs and ) in arbitrary fluorescent units (A.F.U.). Values are shown in box‐and‐whisker plots where each dot is a metastatic lesion and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 8–16 metastatic lesions from 2 to 4 brains per condition, two independent staining with different brains were performed). P value was calculated using two‐tailed t ‐test. C Quantification of percentage of nuclear DDA1 + BB + cells shown in (Fig ). Values are shown in box‐and‐whisker plots where each dot is a metastatic lesion, and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles, and the whiskers go from the minimum to the maximum value ( n = 16 metastatic lesions from 4 brains per condition, 2 independent staining with different brains were performed). P value was calculated using two‐tailed t ‐test. D Representative images showing p‐ERK levels in organotypic cultures from (Fig ). This result was reproduced in three independent staining with organotypic cultures from different mice. Scale bar: 20 µm. E–H Kaplan‐Meier curves showing significant correlation between worse survival post‐brain metastasis and high gene expression levels of AHR (E), DDA1 (F), UBE4B (G), and GPATCH8 (H) in a cohort of 45 breast cancer brain metastasis patients. I, J Distribution of poor prognosis breast cancer subtypes HER2 + and TNBC within the low and high gene expression level cohorts considering the signature (I) or individual genes (J). K, L Quantification of ex vivo BLI of brains (K) and thoracic regions (L) of mice inoculated with H2030‐BrM cells carrying shControl or sh AHR #2 at the endpoint of the experiment (5 weeks after injection of cancer cells). Values are shown in box‐and‐whisker plots where every dot represents a different animal and the line in the box corresponds to the median. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value ( n = 10 shControl mice and n = 10 sh AHR #2 mice). P value was calculated using two‐tailed t ‐test. M Representative sections of brains from shControl and sh AHR #1 mice 5 weeks (experimental endpoint) after intracardiac inoculation of cancer cells. The dotted lines surround the metastases (GFP + ). Scale bar: 1 mm. N Quantification of metastases found in brains inoculated with H2030‐BrM cells with sh AHR . Relative metastatic load was normalized to the respective control. Values are shown in dot plots where every dot represents a different brain and the dotted line corresponds to the mean ± s.e.m. ( n = 8 shControl; n = 5 sh AHR #1; n = 3 sh AHR #2 mice). P value was calculated using two‐tailed t ‐test. O, Q H‐score analysis of AHR (O) and DDA1 (R) in primary tumors with (red) or without (gray) associated relapse. Values are shown in a scattered plot where each dot is a primary tumor and the line corresponds to the median ( n = 100/103 primary tumors with relapse; n = 101/103 primary tumors without relapse, respectively). P value was calculated using two‐tailed t ‐test. P, R Kaplan‐Meier curve comparing relapse‐free survival of primary tumors with high and low values of AHR (P) and DDA1 (R). P value was calculated using log‐rank (Mantel‐Cox) test.

Article Snippet: Slides were incubated with the corresponding primary antibodies as follows: HSP90α/β (clone F‐8, 1:3,000; sc‐13119, Santa Cruz Biotechnology, 8 min), DDA1 (1:2,400; 14995‐1‐AP, ProteinTech), UBE4B (1:500; ab97697, Abcam) and AHR (1:400; 031714, USBiologicals Life Sciences).

Techniques: Generated, Staining, Whisker Assay, Two Tailed Test, Gene Expression, Ex Vivo, Injection, Control