Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: High-content screening using Actin CytoFRET identifies deubiquitinase inhibitors as novel modulators of actin remodeling. (A) Actin CytoFRET principle: Actin-FRET (ACT) cell line expresses EGFP-actin and mOrange-actin fluorescent proteins (FPs). FRET signals are emitted in FRET-ACT cells when EGFP-actin and mOrange-actin locate close to each other in actin filaments. FRET-control (CT) cell line expresses EGFP-actin and mOrange alone that cannot be incorporated into actin filaments, preventing FRET signal emission. FRET signals are monitored by flow cytometry using a ratiometric method. (B) Heatmap representation of Actin CytoFRET screening of a small library of compounds affecting post-translational modifications. Each compound was tested at three concentrations (5, 10, 30 µM) for 60 min on ACT and CT reporter cell lines and FRET signals were recorded by flow cytometry. Data are expressed as the percentage of FRET efficiency between DMSO-treated and compound-treated cells. Jasplakinolid and Latrunculin B were tested as controls for actin polymerization and depolymerization, respectively. (C) Dose-response determination of the activity of DUBi WP1130, b-AP15, PR-619 and VLX1570 on actin FRET efficiency. Data represent the mean ± SD of n = 3 independent experiments. EC50 for each drug were calculated using GraphPad Prism software.

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: High Content Screening, Control, Flow Cytometry, Activity Assay, Software

Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: DUB inhibition increases actin polymerization and blocks leukemic cell chemotactic migration. (A) Confocal microscopy analysis of actin-mOrange distribution in ACT Jurkat cells treated with DMSO or 15 µM of WP1130 and b-AP15 for 30 min. Nuclei were counterstained with DAPI before analysis. Scale bar, 5 µm. (B) Effects of WP1130, b-AP15, PR-619 and VLX1570 on CXCL12-induced Jurkat T cell migration. Cells were migrated against 100 ng/ml of CXCL12 for 90 min in the presence or not of 15 µM of the indicated compound. Results are expressed as the fold of cell migration compared to DMSO control condition. Scatter plots show the mean of n = 3 independent experiments. *** p < 0.001, unpaired t -tests between DMSO and each treated condition. (C) Real time analysis of actin FRET signals induced by 100 ng/ml CXCL12 on ACT cell line treated with DMSO or 15 µM WP1130 and b-AP15 for 30 min. Flow cytometry histograms show representative data on ACT cell line treated with DMSO or DUBs inhibitors. Data are representative of n = 3 independent experiments. The effect of CXCR4 inhibitor AMD3100 (5 µM) on CXCL12-induced actin FRET signals is shown as control.

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Migration, Confocal Microscopy, Control, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: DUB inhibition induces cofilin dephosphorylation. (A) Analysis of actin FRET efficiencies on ACT cell line pre-treated with 500 nM Latrunculin B or 5 µM CK-666 for 1 h prior addition of 15 µM of WP1130 or b-AP15 for one additional hour. Data are expressed as the percentage of FRET efficiency between DMSO-treated and compound-treated cells. Graphs are mean ± SD of n = 3 independent experiments. (B) Immunoblot analysis showing the effect of WP1130 and b-AP15 (1 h, 15 µM) on the levels of actin sequestering proteins. Membranes were probed with the indicated antibodies and anti-ERK2 as loading control. (C) Effect of increasing amounts of WP1130 and b-AP15 on the phosphorylation of cofilin at Ser3. Membranes were probed with the indicated antibodies and quantified against total cofilin signal (bar graph). Upper panel, bar graphs show actin FRET efficiencies recorded on ACT cells upon the same treatment (mean ± SD of 3 independent experiments). immunoblot analysis of lysates obtained from cells treated as above. (D) Time-course analysis of cofilin Ser3 phosphorylation following treatment with 15 µM of WP1130 and b-AP15 for the indicated time. Membranes were probed with the indicated antibodies and quantified against total cofilin signal (bar graph). Upper panel, the graph shows the corresponding effects on actin FRET efficiencies. (E) WP1130 and b-AP15, but not 8TQ, HBX41108 and MG132, reduce cofilin Ser3 phosphorylation levels. Immunoblotting were performed on lysates from Jurkat cells treated with 15 µM of the indicated compound for 30 min. (F) Immunoblot analysis of cofilin Ser3 phosphorylation on cells treated with 500 nM Latrunculin B or 5 µM CK-666 prior addition of 15 µM of WP1130 and b-AP15 for 30 min.

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: Inhibition, De-Phosphorylation Assay, Western Blot, Control, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: Effect of cofilin knockdown on DUBi-induced actin reorganization. (A) Immunoblot analysis showing cofilin downregulation on Jurkat ACT cell line following stable transduction with shCtrl or shCofilin vectors. ERK2, loading control. Densitometric quantification to ERK2 signal is shown (lower panel). (B) Basal actin FRET measurement expressing shCtrl or shCofilin vectors. Data are representative of n = 5 independent experiments. (C) Analysis of actin FRET efficiency following DUB inhibition on shCtrl and shCofilin ACT cell lines. Cells were treated or not with WP1130 and b-AP15 (15 µM each) for 30 min and FRET efficiencies were recorded by flow cytometry. Data are expressed as the percentage of FRET efficiency between DMSO-treated and compound-treated cells. Scatter plots show the mean of n = 5 independent experiments. *** p < 0.001, unpaired t -tests between DMSO and each treated condition. (D) Confocal microscopy analysis of actin-mOrange reorganization in shCtrl and shCofilin ACT cells. Scale bar, 20 µm. (E) Effect of WP1130 and b-AP15 on SSH1 expression and LIMK1/2 and Cofilin phosphorylation. Cells were treated for 1 h at 37°C with the indicated compound (15 µM) and lysates were analyzed by immunoblotting with the indicated antibodies and quantified by densitometry analysis against total cofilin signal (right panel).

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: Knockdown, Western Blot, Transduction, Control, Expressing, Inhibition, Flow Cytometry, Confocal Microscopy, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: Actin cytoskeleton reorganization induced by WP1130 and b-AP15 is ROS dependent. (A) Heatmap representation of actin FRET efficiencies on ACT reporter cells that have been pre-treated with the indicated compounds, following exposure to 15 µM WP1130 or b-AP15 for 60 min. FRET signals were recorded by flow cytometry. Data are expressed as the percentage of FRET efficiency between DMSO-treated and compound-treated cells and are representative of n = 3 independent experiments. (B) Effect of WP1130 and b-AP15 on ROS production. ACT reporter cells that have been pre-treated with 1 mM DTT and 100 µM NAC were incubated with 15 µM WP1130 or b-AP15 for 60 min. Following treatment, cells were stained with 5 µM CM-H 2 DCFDA and analyzed by flow cytometry. Bar graphs show the mean fluorescence intensity (MFI) of ROS staining of n = 3 independent experiments. (C) Whole cell lysates of Jurkat pre-treated with 100 µM NAC before addition or not of 15 µM WP1130 or b-AP15 for 30 min were analyzed by immunoblotting with antibodies against phosphorylated cofilin and total cofilin. (D) Effect of WP1130, b-AP15 and Auranofin on chemotactic migration of leukemic cells pretreated or not with DTT and NAC. Cells were migrated against 100 ng/ml of CXCL12 for 90 min in the presence or not of the indicated compound. Results are expressed as the fold of cell migration compared to the DMSO condition. Bar graphs show the mean of n = 3 independent experiments. *** p < 0.001, unpaired t -tests between DMSO and each treated condition. (E) Effect of Auranofin on ROS production. (F) Dose response curve of the activity of Auranofin on actin FRET efficiency. (G) Immunoblot analysis showing the effect of Auranofin on the phosphorylation of cofilin at Ser3. Densitometric quantification against total cofilin signal is shown (lower panel).

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: Flow Cytometry, Incubation, Staining, Fluorescence, Western Blot, Migration, Activity Assay, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Deubiquitinase Inhibitors Impair Leukemic Cell Migration Through Cofilin Oxidation and Alteration of Actin Reorganization

doi: 10.3389/fphar.2021.778216

Figure Lengend Snippet: Inhibition of DUBs in leukemic cells induces Cofilin oxidation and oligomerization. (A) Jurkat cells stably expressing GFP-cofilin were treated or not with 15 µM WP1130 or b-AP15 for 30 min and lysed. Cell lysates were incubated with anti-GFP nanobody (GFP-Trap) coupled to agarose beads. The trapped proteins and a fraction of the cell lysates (Input) were incubated with DNPH for 15 min at room temperature before immunoblot with antibodies against DNP or GFP. Anti-DNP immunoblot of DNP-labelled oxidized BSA (40 ng) is shown as a control. Data are representative of n = 2 experiments. Densitometric quantification of cofilin oxidation against GFP signal is shown as bar graph (bottom panel). (B) Immunofluorescence microscopy analysis of actin-mOrange and Cofilin distribution in ACT Jurkat cell treated with DMSO or 15 µM of WP1130 and b-AP15 for 30 min. Scale bar, 10 µm. Inset, white arrows show Cofilin aggregates. (C) Schematic overview of the effect of DUB inhibition on cofilin activity, actin polymerization and cell migration.

Article Snippet: Antibodies against β-actin (C4) were purchased from Santa Cruz Biotechnology, cofilin (D3F9), ERK2 (9108), profilin (C56B8), ubiquitin (P4D1), and phospho-ADF/cofilin (3312) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Stable Transfection, Expressing, Incubation, Western Blot, Control, Immunofluorescence, Microscopy, Activity Assay, Migration

Journal: Molecules

Article Title: The Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′- dimethylchalcone from Cleistocalyx operculatus Buds on Human Pancreatic Cancer Cell Lines

doi: 10.3390/molecules24142538

Figure Lengend Snippet: Effect of DMC on caspase activation and PARP degradation protein expression in PANC-1 cells. Cells were treated with DMC (3–30 µM) for 48 h. Protein 50 µg/lane from cells lysates were electrophoresed on SDS-PAGE gels, then transferred to total blot PVDF membranes. β-Actin was used as a control, (–), 0.1% DMSO-treated cells. The experiments were carried out in three replicates.

Article Snippet: Protein contents were normalized by reprobing the same membrane with anti-β-actin detection; previously used membranes were soaked in stripping buffer (Gene Bio-Application Ltd., Yavne, Israel) at room temperature for 20 min.

Techniques: Activation Assay, Expressing, SDS Page

Journal: Molecules

Article Title: The Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′- dimethylchalcone from Cleistocalyx operculatus Buds on Human Pancreatic Cancer Cell Lines

doi: 10.3390/molecules24142538

Figure Lengend Snippet: Effect of DMC on Bcl-2, Bax and Cyt-c protein expression in PANC-1 cells. Cells were treated with DMC (3–30 µM) for 48 h. Protein 50 µg/lane from cells lysates were electrophoresed on SDS-PAGE gels, then transferred to total blot PVDF membranes. β-Actin was used as a control, (–) 0.1% DMSO-treated cells. The experiments were carried out in three replicates. * P < 0.05 and P < 0.01 compared with control group.

Article Snippet: Protein contents were normalized by reprobing the same membrane with anti-β-actin detection; previously used membranes were soaked in stripping buffer (Gene Bio-Application Ltd., Yavne, Israel) at room temperature for 20 min.

Techniques: Expressing, SDS Page

Journal: Journal of Medicinal Chemistry

Article Title: Structural Fine-Tuning of Desmuramylpeptide NOD2 Agonists Defines Their In Vivo Adjuvant Activity

doi: 10.1021/acs.jmedchem.1c00644

Figure Lengend Snippet: Effects of the MDP and desmuramylpeptide treatments on the release of cytokines from human PBMCs. Cytokine concentrations were measured after 18 h stimulation with MDP (2 μM) or the desmuramylpeptides (2 μM) in the absence or presence of LPS (10 ng/mL). Data are expressed as mean ± SEM of 4 independent experiments. # p < 0.05, ### p < 0.001 versus untreated controls; * p < 0.05, ** p < 0.01, *** p < 0.001 versus LPS-treated PBMCs.

Article Snippet: Cytokine production was determined with the BD Cytometric Bead Array Human Inflammatory Cytokines Kit (contents: IL-8, IL-1β, IL-6, IL-10, TNF, IL-12p70; BD Bioscience) on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.).

Techniques: