Journal: Frontiers in Veterinary Science

Article Title: Abemaciclib induces G1 arrest and lysosomal dysfunction in canine melanoma cells: synergistic effects with fenbendazole

doi: 10.3389/fvets.2025.1603686

Figure Lengend Snippet: Dextran uptake assay and vacuolization inhibition test with V-ATPase inhibitor and protein synthesis inhibitor. (A) Dextran uptake assay. Under serum starvation conditions, CMeC1 control cells exhibited uptake of FITC-Dextran (green) through pinocytosis. However, treatment with 2.5 μM abemaciclib led to vacuole formation in the cytoplasm, but these vacuoles did not contain FITC-Dextran, suggesting that the vacuoles originated from intracellular components rather than extracellular uptake. Nuclei were stained with DAPI (blue). Images include bright-field (BF), DAPI, FITC-Dextran, DAPI merged with FITC, and BF merged with DAPI and FITC. Scale bar = 50 μm. (B) Vacuolization inhibition test with V-ATPase inhibitors (bafilomycin A1 and concanamycin A) and a protein synthesis inhibitor (cycloheximide). Cells were pre-treated with inhibitors (bafilomycin A1 20 nM, concanamycin A 10 nM, or cycloheximide 2 μM) for 2 h prior to abemaciclib treatment and observed after 12 h. Bafilomycin A1 and concanamycin A effectively suppressed vacuolization at 12 h. Cycloheximide partially suppressed vacuole formation at 12 h. Scale bar = 50 μm. BF, bright field.

Article Snippet: To investigate the origin of vacuoles, bafilomycin A1 (MedChemExpress, Monmouth Junction, NJ, United States; Cat. #HY-100558), concanamycin A (MedChemExpress; Cat. #HY-N1724), or cycloheximide (Tocris Bioscience, Bristol, United Kingdom; Cat. #0970) was added 2 h prior to abemaciclib treatment and co-incubated with 2.5 μM abemaciclib for 12 h. Images were captured using a phase-contrast microscope.

Techniques: Inhibition, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)

doi: 10.3390/ijms25168984

Figure Lengend Snippet: Real-time HiBiT detection assays to measure CCR2-HaloTag-HiBiT levels over 24 h, after treatment with indicated compounds. ( A ) Schematic drawing of the real-time HiBiT assays. HEK293-LgBiT cells were transfected with 3 µg of CCR2-HaloTag HiBiT (unless indicated otherwise), allowing the immediate complementation of HiBiT and LgBiT to form Nanoluciferase (NanoLuc). Transfected cells were then treated with selected compounds and luminescence, expressed as relative light units (RLUs), and continuously measured over 24 h. ( B ) Kinetic degradation profiles obtained in HEK293-LgBiT cells transfected with 1 µg, 3 µg, or 5 µg of CCR2-HaloTag HiBiT after treatment with 1 µM of HaloPROTAC3. ( C , D ) Kinetic degradation profiles of CCR2-HaloTag HiBiT after treatment with multiple concentrations of HaloPROTAC3 ( C ) or ent -HaloPROTAC3 ( D ). ( E ) Degradation curves showing the fractional RLU values corresponding to D max at each concentration of the compound. D max values were obtained from the real-times traces shown in ( C , D ). ( F ) Kinetic profiles of CCR2-HaloTag HiBiT after treatment with a single concentration (1 µM) of the indicated compounds. ( G ) Kinetic degradation profiles of CCR2-HaloTag HiBiT after treatment with 10 µM of cycloheximide or 1 µM of HaloPROTAC3 in the absence or presence of cycloheximide. In all cases, luminescence (RLU) was measured over 24 h in 15-min intervals. RLU values from mock-transfected HEK293-LgBiT cells were used for baseline-correction, and data were normalized to vehicle control. Data are shown as mean ± SEM of at least three independent experiments performed in triplicates. Statistical differences between fractional RLU values of compounds versus ent -HaloPROTAC3 ( F ) were analyzed using one-way ANOVA with Dunnett’s post-hoc test: *** p < 0.001, **** p < 0.0001.

Article Snippet: VL285, Bortezomib (PS-341), Pevonedistat (MLN4924), MG-132, Chloroquine diphosphate, Bafilomycin A1 (Baf-A1) were all purchased from Selleck Chemicals (Bio-Connect, Huissen, The Netherlands); Cycloheximide from LKT Labs (St. Paul, MN, USA); and chemokine ligand CCL2 from PeproTech (Cranbury, NJ, USA).

Techniques: Transfection, Concentration Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Inducing Receptor Degradation as a Novel Approach to Target CC Chemokine Receptor 2 (CCR2)

doi: 10.3390/ijms25168984

Figure Lengend Snippet: Lytic HiBiT detection assays to measure CCR2 levels in HEK293T cells transfected with 5 µg of CCR2-HaloTag HiBiT. Transfected cells were pretreated with the indicated inhibitors for 2 h before treatment with vehicle ( A , C , E ) or 1 µM of HaloPROTAC3 ( B , D , F ) for three more hours and measuring luminescence. E3 ligase inhibitors VL285, pomalidomide and idasanutlin are shown in blue ( A , B ); proteasomal (MG-132 and bortezomib) and neddylation (MLN-4924) inhibitors in magenta ( C , D ); lysosomal inhibitors bafilomycin-A1 and chloroquine in red; and the protein synthesis inhibitor cycloheximide in green ( E , F ). RLU values from mock-transfected cells were used for baseline-correction in all cases. Graphs show mean ± SEM values obtained from at least three independent experiments performed in triplicate. Statistical differences between normalized RLU values were analyzed using one-way ANOVA with Dunnett’s post-hoc test: ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: VL285, Bortezomib (PS-341), Pevonedistat (MLN4924), MG-132, Chloroquine diphosphate, Bafilomycin A1 (Baf-A1) were all purchased from Selleck Chemicals (Bio-Connect, Huissen, The Netherlands); Cycloheximide from LKT Labs (St. Paul, MN, USA); and chemokine ligand CCL2 from PeproTech (Cranbury, NJ, USA).

Techniques: Transfection

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: ( A ) Itgb1 mRNA levels in WT and USP12/46-dKO fibroblasts determined by qPCR. Statistical analysis was carried out by RM one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-dKO cl1 or cl2 fibroblasts ( P = 0.1278 and 0.0178, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( B , C ) WB ( B ) and densitometric quantification ( C ) of Itgb1 protein levels in WT and USP12/46-dKO fibroblasts at indicated time points after cycloheximide (CHX) treatment (5 μg/ml). Gapdh served as a loading control. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-dKO cl1 or cl2 fibroblasts at the 24-hour time point ( P = 0.0327 and 0.0127, respectively). * P < 0.05. Data were shown as Mean ± SD, n = 3 independent experiments. ( D ) Quantification of surface Itgb1 degradation kinetics in WT and USP12/46-dKO fibroblasts. The amount of Itgb1 remaining over indicated times were measured by capture-ELISA. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts with USP12/46-dKO cl1 or cl2 fibroblasts at the 24-h time point (both P < 0.0001). **** P < 0.0001. Data were shown as Mean ± SD, n = 3 independent experiments. ( E ) Quantification of surface Itgb1 degradation kinetics in WT fibroblasts stably expressing EGFP, and USP12/46-dKO fibroblasts stably expressing EGFP-USP12 WT or EGFP-USP12 C48S determined by capture-ELISA. Statistical analysis was carried out by ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing the WT fibroblasts expressing EGFP with USP12/46-dKO fibroblasts expressing EGFP-USP12 WT or EGFP-USP12 C48S ( P = 0.9983 and 0.0248, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( F , G ) The internalization rate ( F ) and the recycling rate ( G ) of Itgb1 in USP12/46-dKO fibroblasts stably expressing EGFP-USP12 WT or EGFP-USP12 C48S determined by capture-ELISA. Statistical analysis was carried out by two-sided Welch’s t -test comparing USP12/46-dKO fibroblasts expressing EGFP-USP12 WT or EGFP-USP12 C48S at the end time point. The P values in ( F ) and ( G ) are 0.6734 and 0.0168, respectively. * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( H , I ) WB ( H ) with densitometric quantification ( I ) of Itgb1 protein levels in lysates of WT and USP12/46-dKO fibroblasts treated with DMSO, MG132 (0.5 uM) or BafA1 (40 nM) for 9 h. Gapdh served as a loading control. Statistical analysis was carried out by RM two-way ANOVA with Dunnett’s multiple comparison test comparing the DSMO group with MG132 or BafA1 group in WT fibroblasts ( P = 0.3185 and 0.0151, respectively); in USP12/46-dKO cl1 fibroblasts ( P = 0.2362 and 0.0195, respectively); and in USP12/46-dKO cl2 fibroblasts ( P = 0.1809 and 0.0344, respectively). * P < 0.05; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( J ) Itgb1 surface levels in WT, USP12/46-dKO, and SNX17-KO fibroblasts treated with DMSO or BafA1 (40 nM) for 9 h determined by flow cytometry. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test comparing DSMO group with BafA1 group in WT fibroblasts ( P = 0.0036); in USP12/46-dKO cl1 fibroblasts ( P = 0.7237); in USP12/46-dKO cl2 fibroblasts ( P = 0.0041); and in SNX17-KO fibroblasts ( P < 0.0001). ** P < 0.01; **** P < 0.0001; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments. ( K ) Representative immunofluorescence (IF) images of Itgb1 and Lamp1 in WT and USP12/46-dKO fibroblasts treated with DMSO or BafA1 (100 nM) for 3 h. White/pink arrowheads show the accumulation of Itgb1 in Lamp1-positive endo/lysosomes. Boxes indicate magnified cell regions displayed in the Zoom panel. Sum intensity projections from confocal stacks are presented. Scale bar, 10 µm. ( L ) Superplots showing the Pearson correlation coefficients (PCC) between Itgb1 and Lamp1 in WT and USP12/46-dKO fibroblasts. Statistical analysis was carried out by RM two-way ANOVA with Šidák’s multiple comparison test comparing DMSO-treated WT fibroblasts with BafA1-treated WT fibroblasts ( P = 0.0047); DMSO-treated WT fibroblasts with DMSO-treated USP12/46-dKO fibroblasts ( P = 0.1903); BafA1-treated WT fibroblasts with BafA1-treated USP12/46-dKO fibroblasts ( P = 0.0019); DMSO-treated USP12/46-dKO fibroblasts with BafA1-treated USP12/46-dKO fibroblasts ( P = 0.0002); ** P < 0.01; *** P < 0.001; n.s. not significant. Data were shown as Mean ± SD, n = 3 independent experiments; 49 cells were analyzed in DMSO-treated WT cells, 43 in BafA1-treated WT cells, 52 in DMSO-treated USP12/46-dKO cells, and 45 in BafA1-treated USP12/46-dKO cells. .

Article Snippet: Cycloheximide , Santa Cruz Biotechnology , sc-3508A.

Techniques: Comparison, Control, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Flow Cytometry, Immunofluorescence

Journal: EMBO Reports

Article Title: The USP12/46 deubiquitinases protect integrins from ESCRT-mediated lysosomal degradation

doi: 10.1038/s44319-024-00300-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Cycloheximide , Santa Cruz Biotechnology , sc-3508A.

Techniques: Recombinant, Expressing, Sequencing, Real-time Polymerase Chain Reaction, Protease Inhibitor, cDNA Synthesis, SYBR Green Assay, BIA-KA, Western Blot, Software, Microscopy

Journal: STAR Protocols

Article Title: Integrated workflow for discovery of microprotein-coding small open reading frames

doi: 10.1016/j.xpro.2023.102649

Figure Lengend Snippet:

Article Snippet: Cycloheximide (CHX) , Thermo Fisher , Cat# J66004XF.

Techniques: Recombinant, Saline, Protease Inhibitor, Sterility, Staining, Purification, Sequencing, Spectrophotometry, Reverse Transcription, Software