Journal: Cell Reports Medicine

Article Title: Pharmacological Activation of Non-canonical NF-κB Signaling Activates Latent HIV-1 Reservoirs In Vivo

doi: 10.1016/j.xcrm.2020.100037

Figure Lengend Snippet: Ciapavir Reverses HIV-1 Latency with Improved Potency and Efficacy (A) Structures of seven commercially available Smac mimetics that were tested for LRA activity. (B) 2D10 Jurkat cells were incubated with SBI-0637142 and seven additional Smac mimetic compounds for 48 h. Latency reversal was assessed by measuring GFP expression by flow cytometry. Baseline activation levels are indicated by dashed line. Data represent mean and SD of two biological replicates (n = 2). # BV-6 could not be assessed at a concentration of 20 μM due to cytotoxicity (see Figure S1 A). (C) cIAP1 degradation after Smac mimetic treatment of 2D10 cells for 24 h was evaluated by automated capillary western blot (SimpleWestern) analysis. (D) Structure of the monovalent compound SBI-0637142 and the optimized bivalent Smac mimetic Ciapavir. (E) 2D10 cells were incubated with increasing concentrations of SBI-0637142 and Ciapavir for 48 h. Data represent mean and SD of three experiments. Difference between compound LRA activities is significant at concentrations greater than 0.5 nM (p < 0.05; determined by two-way ANOVA; n = 3). (F) The LRA activity of SBI-0637142 and Ciapavir is dependent on the NIK signaling axis. 2D10 cells, unmodified (wt) and with a deletion of the NIK gene (ΔNIK), were treated with 1 μM SBI-0637142, 1 μM Ciapavir, or 10 nM bryostatin-1 for 48 h. Data represent mean and SD of three biological replicates. Statistical significance was analyzed by two-way ANOVA with Dunnett’s multiple comparison test (n = 3). (G) Ciapavir acts synergistically with JQ1 and I-BET151 to reverse HIV latency. 2D10 cells were treated with compound combinations for 48 h prior to analysis of cell viability and LRA activity (see Figure S1 B). Synergy is shown as excess over Bliss (EOB). Values greater than 0 indicate positive synergy of the compounds. Gray fields indicate cell viability <70%. Heatmaps represent average values of two experiments.

Article Snippet: Western blot analysis was performed on a Peggy Sue Automated Western Blot System (ProteinSimple) with sample concentrations adjusted to 1.8 μg/μL and using primary antibodies against cIAP1 (AF8181, R&D Systems) at a concentration of 5 μg/mL, and against GAPDH (AF5718, R&D Systems) at a concentration of 0.04 μg/mL as loading control.

Techniques: Activity Assay, Incubation, Expressing, Flow Cytometry, Activation Assay, Concentration Assay, Western Blot, Comparison

Journal: Cell Reports Medicine

Article Title: Pharmacological Activation of Non-canonical NF-κB Signaling Activates Latent HIV-1 Reservoirs In Vivo

doi: 10.1016/j.xcrm.2020.100037

Figure Lengend Snippet: Ciapavir-Mediated Sustained Target Engagement In Vivo in Mice (A) Plasma exposure of SBI-0637142 and Ciapavir after 10 and 20 mg/kg intraperitoneal dosage at 2 h. Error bars represent mean ± SD with n = 2 (SBI-0637142) and n = 3 (Ciapavir) in each group. (B) Western blot showing cIAP1/2 degradation in spleen and thymus 2 h after treatment. Samples of two mice are shown for each condition. (C) Pharmacokinetic time course of Ciapavir in mice after 10 mg/kg dosed intraperitoneally (n = 3; geometric mean ± geometric SD). Additional PK parameters are detailed in . (D) Ciapavir treatment leads to sustained cIAP1/2 degradation over 24 h. cIAP1 levels in spleen and thymus of mice treated with 10 mg/kg Ciapavir by i.p. dosing for the indicated amount of time were analyzed by western blot. Samples of two mice are shown for each condition. (E) C57BL/6 mice were dosed (i.p.) with 10 and 20 mg/kg Ciapavir. 2 mg/kg LPS served as positive control. Serum samples were isolated 2 and 24 h after compound administration and analyzed for cytokine levels. Graphs show mean and SD of four animals. Symbols represent values of individual animals. Detection limit is indicated by dotted line. Log transformed data were analyzed with a two-way ANOVA using Dunnett’s multiple comparison correction (n = 4).

Article Snippet: Western blot analysis was performed on a Peggy Sue Automated Western Blot System (ProteinSimple) with sample concentrations adjusted to 1.8 μg/μL and using primary antibodies against cIAP1 (AF8181, R&D Systems) at a concentration of 5 μg/mL, and against GAPDH (AF5718, R&D Systems) at a concentration of 0.04 μg/mL as loading control.

Techniques: Drug discovery, In Vivo, Clinical Proteomics, Western Blot, Positive Control, Isolation, Transformation Assay, Comparison

Journal: Cell Reports Medicine

Article Title: Pharmacological Activation of Non-canonical NF-κB Signaling Activates Latent HIV-1 Reservoirs In Vivo

doi: 10.1016/j.xcrm.2020.100037

Figure Lengend Snippet:

Article Snippet: Western blot analysis was performed on a Peggy Sue Automated Western Blot System (ProteinSimple) with sample concentrations adjusted to 1.8 μg/μL and using primary antibodies against cIAP1 (AF8181, R&D Systems) at a concentration of 5 μg/mL, and against GAPDH (AF5718, R&D Systems) at a concentration of 0.04 μg/mL as loading control.

Techniques: Virus, Modification, Recombinant, Viability Assay, Multiplex Assay, Digital PCR, Software, Cell Isolation

Journal: Stem cells (Dayton, Ohio)

Article Title: Inhibitor of Apoptosis Proteins Determines Glioblastoma Stem-Like Cell Fate in an Oxygen-Dependent Manner.

doi: 10.1002/stem.2997

Figure Lengend Snippet: Figure 1. Apoptosis inhibitor proteins (IAPs) distribution between peri-necrotic and non-necrotic areas in human glioblastomas (GBMs) and set up of in vitro hypoxic condition. (A): cIAP1, cIAP2, XIAP, and ML-IAP protein expression was analyzed by immunohistochemistry on serial slides of FFPE samples from eight human GBMs. Carboxyanhydrase IX stainings highlight hypoxic areas and asterisk represents palisading cells. Pictures of a representative sample are shown. Scale bar: 100 μm. (B): Scatter plots represent cIAP1, cIAP2, XIAP, and ML-IAP mRNA expression in palisading cells and common tumor cells of human GBMs. Median expression is represented by the horizon- tal red line and mean expression by the red cross. Data were obtained from NCBI GEO. (C): Adrenomedullin (ADM) mRNA was quantified in palisading cells and in common tumor cells. Median expression is represented by the horizontal red line and mean expression by the red cross. Data were obtained from NCBI GEO. (D): GBM9 cells were grown in monolayer in normoxia or in hypoxia for 8 days. ADM mRNA level was analyzed by Q-RT-PCR and fold increase ADM mRNA level is shown + SEM (n = 3 independent experiments); *, p < .05. (E): cIAP1, cIAP2, XIAP, and ML-IAP mRNA levels of GBM9 cells (grown in monolayer) were analyzed by Q-RT-PCR in normoxia or in hyp- oxia and fold increase mRNA levels is shown + SEM (n = 3 independent experiments). Abbreviation: ns, not significant.

Article Snippet: After steam-heat-induced antigen retrieval, 5-μm sections of formalin-fixed paraffin-embedded samples were tested for the presence of cIAP1 (AF8181, R&D Systems, Wiesbaden, Germany), cIAP2 (AF8171, R&D Systems), XIAP (clone 48, BD Biosciences, Franklin Lake, NJ), ML-IAP (IMG-347A, Imegenex, Cambridge, U.K.), carbonic anhydrase IX (CAIX; GTX15086, GeneTex, Inc., Irvine).

Techniques: In Vitro, Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 stabilizes and stimulates E2F1 in an E3-ubiquitin ligase activity-dependent manner. ( a, b and d ) E2F1 activity and expression in Hela cells ( a and b ) or in cIAP1 −/− /cIAP2 −/− MEFs ( d ) transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-3HA-E2F1, along with empty vector (Vector), cIAP1, cIAP1-H588A (devoid of E3-ubiquitin ligase activity) or cIAP1-F616A (lacking dimerization capacity) mutant encoding vector. HeLa cells were treated for 24 h with PYR-41 10–35 μ M before analysis ( a ). Upper panels: E2F1 transcriptional activity was assessed in gene luciferase experiments. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1 alone. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t -test. *** P <0.001, **0.001< P <0.01, *0.01< P <0.1, NS P >0.1. Lower panels: the expression of the constructs was analyzed by a western blot analysis. β -Actin or HSC70 are used as loading control. *Unspecific bands. ( c ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1, His-Ubiquitin encoding vectors, with an empty vector, cIAP1 or cIAP1 F616A (F/A) mutant constructs. Ubiquitinated proteins were pulled-down by using non-selective or K63-TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody. ( e ) Western blot analysis of cIAP1 or 3HA-E2F1 expression in HeLa cells transfected with pCMV-3HA-E2F1 and with empty vector (Vector), cIAP1 or cIAP1-F616A mutant (dimerization defective mutant) encoding vector, treated or not with MG132 5 μ M overnight. ( f ) In vitro ubiquitination assay of GST-E2F1 fusion protein immobilized on gluthatione sepharose and incubated with ubiquitin, E1 and E2 recombinant proteins with or without recombinant cIAP1

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Western Blot, Control, In Vitro, Incubation, Recombinant

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 overexpression revealed a K63 ubiquitination of E2F1 on lysine residue 161/164. ( a ) Schematic representation of E2F1 protein structure indicating the lysine residues and lysine clusters. ( a ) Cyclin A: CDK2-binding domain; DBD: DNA-binding domain, dimer: dimerization domain; MB: marked box: Rb: Rb-binding domain. ( b ) Gene luciferase experiments performed in HeLa cells transfected with CCNE promoter-Firefly luciferase reporter plasmid, pCMV-HA (vector), HA-tagged-E2F1 (one single or 3HA) or HA-E2F1 mutants in which indicated K have been mutated into R along with empty vector (vector) or cIAP1-encoding vector. Luciferase activity was normalized to β -galactosidase activity and expressed as fold induction of promoter stimulated by E2F1. Mean±S.D. of at least three independent experiments. Statistical analysis performed using Student’s t test. *** P <0.001, *0.01< P <0.1, NS P >0.1. The expression of the E2F1 constructs was checked by a western blot analysis (upper panel). β -Actin is used as loading control. ( c and d ) Ubiquitination assay performed in HeLa cells transfected with 3HA-E2F1 wt, K137R mutant or K161/164R mutant, His-Ubiquitin, with an empty vector or cIAP1 or cIAP1 F616A (F/A) mutant constructs. E2F1 is IP by using specific anti-E2F1 antibody and ubiquitination is revealed by using pan-ubiquitin antibody (FK2) ( c ), or ubiquitinated proteins were pulled-down by using K63-specific TUBEs and ubiquitinated E2F1 is revealed by using an anti-E2F1 antibody ( d ). ( e ) IP analysis of the interaction of cIAP1 with E2F1 wt or K161/164 R mutant. Hela cells were transfected with 3HA-E2F1 encoding constructs and cIAP1. cIAP1 or E2F1 were IP using anti-cIAP1 or anti-HA antibody and cIAP1-E2F1 interaction was revealed by a western blot analysis. KO: cell lysate from cIAP1 −/− KO MEFs was used to check nonspecific reactivity of the antibody

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Over Expression, Ubiquitin Proteomics, Residue, Binding Assay, Luciferase, Transfection, Plasmid Preparation, Activity Assay, Expressing, Construct, Western Blot, Control, Mutagenesis

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: cIAP1 is required for DNA damage-induced stabilization of E2F1. ( a ) Western blot analysis of E2F1 and cIAP1 in U2OS cells transfected with control or cIAP1-siRNA and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells transfected with cIAP1 siRNA or cIAP1-encoding construct and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes and the efficiency of siRNA were checked by a western blot analysis (input). β -Actin was used as loading control. ( c ) Ubiquitination assay performed in HeLa cells transfected with control or cIAP1 siRNA and with 3HA-E2F1 and His-tagged ubiquitin wt. When indicated, cells were treated for 6 h with 10 μ M etoposide. Ubiquitinated proteins were pulled-down by using K63 specific TUBEs and ubiquitinated E2F1 is revealed by using E2F1 antibody. ( d ) Western blot analysis of E2F1 in U2OS cells transfected with 3HA-E2F1 or 3HA-E2F1-K161/164R mutant and treated with indicated concentration of etoposide for 6 h. β -Actin was used as loading control. ( e ) Ubiquitination of E2F1 in HeLa cells transfected with pCMV-3HA-E2F1 or pCMV-3HA-E2F1 K161/164R, and with His-tagged ubiquitin wt, and then treated for 6 h with 10 μ M etoposide. E2F1 was IP using anti-HA antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both untreated samples. ( f ) U2OS cells were transfected with cIAP1 siRNA or treated with 17 nM GDC-0152 for 1 h, and then treated with 10 μ M etoposide for 48 h. tp73 mRNA expression was measured by RT-qPCR. UT: untreated cells. Results were normalized to hprt mRNA and expressed relatively to control untreated cells. Mean±S.D. of three independent experiments

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Western Blot, Transfection, Control, Concentration Assay, Ubiquitin Proteomics, Construct, Expressing, Mutagenesis, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: PRMT-mediated arginine methylation of E2F1 is required for regulation of E2F1 by cIAP1. ( a ) Western blot analysis of E2F1 and cIAP1 in HeLa cells transfected with 3HA-E2F1 and cIAP1-encoding constructs, and treated for 24 h with 100 μ M PRMT inhibitor AMI-1. HSC70 was used as loading control. ( b ) Ubiquitination of E2F1 in HeLa transfected with pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt, and then treated for 16 h with 50 μ M AMI-1±10 μ M etoposide for 6 h. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene is checked by western blot. β -Actin was used as loading control. ( c ) E2F1 transcriptional activity as measured by using a gene reporter luciferase assay performed in Hela cells transfected as in a in the presence of CCNE promoter-Firefly luciferase reporter plasmid and treated with increasing concentration of AMI-1 for 24 h. Results are expressed as average±S.D. from at least three experiments. Statistical analysis performed using Student’s t- test. *** P <0.001, **0.001< P <0.01, NS P >0.1. ( d ) Ubiquitination profile of E2F1 in HeLa transfected with Control (Co), PRMT-1 or PRMT-5-siRNA and pCMV-3HA-E2F1, pCI-cIAP1, and with His-tagged ubiquitin wt. E2F1 was IP using anti-HA antibody and ubiquitin revealed using pan anti-ubiquitin antibody (FK2). The expression of the transgene and the efficiency of siRNA were checked by western blot analysis. β -Actin was used as loading control

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Methylation, Western Blot, Transfection, Construct, Control, Ubiquitin Proteomics, Expressing, Activity Assay, Luciferase, Plasmid Preparation, Concentration Assay

Journal: Cell Death & Disease

Article Title: DNA damage and S phase-dependent E2F1 stabilization requires the cIAP1 E3-ubiquitin ligase and is associated with K63-poly-ubiquitination on lysine 161/164 residues

doi: 10.1038/cddis.2017.222

Figure Lengend Snippet: K63 ubiquitination of E2F1 is cell cycle regulated. ( a ) Western blot analysis of E2F1, cyclin A and cIAP1 in U2OS cells transfected with cyclin A siRNA±cIAP1 siRNA. HSC70 was used as loading control. ( b ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA±cIAP1 siRNA, then 24 h later with pCMV-3HA-E2F1 and His-tagged ubiquitin wt. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. ( c ) Western blot analysis of the expression of 3HA-E2F1 wt or K161/164 R mutant and cyclin A in HeLa cells transfected with cyclin A siRNA. β -Actin was used as loading control. ( d ) Ubiquitination profile of E2F1 in HeLa cells co-transfected with cyclin A siRNA, then 24 h later with pCMV-3HA-E2F1 or the K161/164 R mutant and His-tagged K63-only ubiquitin. E2F1 was IP using anti-E2F1 antibody and ubiquitin revealed using K63-specific ubiquitin chain antibody (K63-Ub). The expression of the transgenes was checked by a western blot analysis. β -Actin was used as loading control. The level of expression of E2F1 constructs has been adjusted in order to get equivalent ubiquitin level in both samples transfected with control siRNA. ( e ) Western blot analysis of cIAP1 E2F1, cyclin E, Rb, phospho (S780, S807/811) Rb in U2OS cells transfected with cIAP1-encoding construct. HSC70 was used as loading control. ( f ) Schematic representation of the regulation of E2F1. In the G1 phase of cell cycle, E2F1 is complexed to Rb. In the S phase, E2F1 is first methylated on arginine residue par PRMT, then K63-ubiquitinated on K161/164 in a cIAP1-dependent manner, leading to stabilization and activation of the protein. In the late S, cyclin A inhibits K63 ubiquitination of E2F1, binds to and phosphorylates E2F1 and promotes UPS-mediated degradation

Article Snippet: For the in vitro ubiquitination assay, GST-E2F1 fusion protein was produced in Escherichia coli by using the pGEX-E2F1 construct, immobilized on gluthatione sepharose beads (GE Healthcare) and incubated Sigma at 37 °C for 1 h with recombinant cIAP1 (R&D System), 100 nM of recombinant human E1 ubiquitin-activating enzyme UBE1 (Boston Biochem, Cambridge, MA, USA), 1.5 μ g of recombinant human E2 enzyme Ubc H5a/UBE2D1 (Boston Biochem), 0.2 mM of recombinant human ubiquitin (R&D Systems) in a buffer containing 75 mM Tris pH 8.2 mM DTT, 5 mM de MgCl 2 , 4 mM ATP.

Techniques: Ubiquitin Proteomics, Western Blot, Transfection, Control, Expressing, Mutagenesis, Construct, Methylation, Residue, Activation Assay