Journal: NPJ Breast Cancer

Article Title: Overexpression of COL11A1 confers tamoxifen resistance in breast cancer

doi: 10.1038/s41523-024-00645-3

Figure Lengend Snippet: a , b Western blotting was used to detect the expression of COL11A1 in MCF-7 and T47D cells treated with indicated concentrations of 4-OHT for 72 h. c , d The protein expression levels of ERα in MCF-7, MCF-7/TamR, T47D, and T47D/TamR cells were evaluated by western blotting and quantified. e , f Western blotting analysis of ERα in MCF-7/TamR and T47D/TamR cells with or without COL11A1 knockdown. g , h MCF-7/TamR and T47D/TamR cells with or without COL11A1 knockdown were incubated with 5 μM 4-OHT for 72 h. The expression of ERα was examined by western blotting, β-Actin was used as a loading control. i qPCR analysis of the ERα mRNA expression in MCF-7/TamR cells with or without COL11A1 knockdown. j MCF-7/TamR cells transfected with the indicated siRNAs were treated with cycloheximide (CHX, 20 μg/ml), and collected at the indicated times for western blot. k qPCR analysis of the mRNA expressions of CCND1, NRIP1, PgR and GREB1 in MCF-7/TamR cells with or without COL11A1 knockdown. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To measure the effect of COL11A1 on ERα protein stability, the MCF-7/TamR cells transfected with the indicated siRNAs were treated with the protein synthesis inhibitor cycloheximide (CHX, #S7418, Selleck) for the indicated durations before collection, and then subjected to western blot analysis.

Techniques: Western Blot, Expressing, Knockdown, Incubation, Control, Transfection

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