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Image Search Results
Journal: Cell reports
Article Title: Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.
doi: 10.1016/j.celrep.2023.112570
Figure Lengend Snippet: Figure 1. RTK activation triggers ERK reactivation and persister development (A) Diagram illustrating BRAFV600E-driven abnormal cell proliferation. (B) Schematic of opto-RTK activation. Opto-RTK consists of the cytoplasmic domain of FGFR1 (aa 398–822) fused to a myristoylation signal peptide and CRY2PHR, which enables homo-oligomerization upon blue light exposure. (C) Normalized ERK activity traces in WM983B cells pretreated with Vem (1 mM) alone or in combination with Cobi (100 nM) or Tram (100 nM) for 24 h before imaging. ERK activity is normalized to prestimulation values. Data represent mean ± 95% CI (n > 50 cells for each condition). (D) Representative images of p-ERK staining (top) and violin plots of p-ERK/t-ERK (bottom). WM983B cells were exposed to the indicated drugs for 24 h before light stimulation for 1 h (n > 1,000 cells for each condition). Scale bar, 20 mm. (E) Immunoblotting for p-MEK staining (top), p-ERK (middle), and the loading control b-actin (bottom). Cells were treated with Vem (1 mM) plus either Cobi (100 nM) or Tram (100 nM) for 24 h before light stimulation for 1 h. (F) Dose-response analysis of p-ERK levels. WM983B cells were treated with Cobi at the indicated concentrations for 24 h and treated with or without light stimulation for 1 h. p-ERK levels are normalized to the lowest concentration and minimum values. Solid line represents the best fit (n = 3 biological replicates). (G) Density scatterplots of Hoechst versus EdU staining, with cell density color coded in gray scale (n = 2,000 cells for each condition). (H and I) Percentage of S-phase cells in WM858 (H) and WM983B (I) cells treated with either Vem (1 mM) alone (left) or Vem combined with Cobi (100 nM) (right). Dotted lines in graphs indicate the day with or without mitogen stimulation. Data represent mean ± SEM (n = 3 biological replicates). p values comparing conditions with and without mitogen stimulation were calculated using two-tailed paired t tests (*p % 0.05, **p % 0.001). See also Figures S1 and S2.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trypsin-EDTA, 0.25% 1X, phenol red, without Calcium and Magnesium Genesee Cat# 25-510 Bovine Serum Albumin (BSA) Protease-free powder Millipore Sigma Cat# A3311 5-Ethynyl-2’-deoxyuridine (EdU) Millipore Sigma Cat# 900584 AFDye 647 Picolyl Azide Click Chemistry Tools Cat# 1300 (+)-SODIUM L-ASCORBATE Millipore Sigma Cat# A4034 COPPER(II) SULFATE (CuSO4) Millipore Sigma Cat# C1297 Halt protease inhibitor cocktail Thermo Scientific Cat# 1861279 PhosSTOP Millipore Sigma Cat# 4906845001 Critical commercial assays Proteome Profiler Human Phospho-RTK Array Kit R&D Systems Cat# ARY001B GeneJET Plasmid Miniprep Kit Thermo Scientific Cat# K0503 NucleoBond Xtra Midi prep kit Macherey-Nagel Cat# 740410.100 Experimental models:
Techniques: Activation Assay, Activity Assay, Imaging, Staining, Western Blot, Control, Concentration Assay, Two Tailed Test
Journal: Cell reports
Article Title: Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.
doi: 10.1016/j.celrep.2023.112570
Figure Lengend Snippet: Figure 2. RTK activation induces more effective ERK activation in persisters than in non-persisters (A) Schematic illustrating kinase-translocation reporters (KTRs) for ERK, CDK4/6, and CDK2 activity. (B) Experimental design to induce and isolate persisters. Persisters were identified based on CDK2 activity 20–35 h after mitogen stimulation (CDK2 activity >0.8 for over 1 h). (C and D) Heatmaps of single-cell traces for ERK (left), CDK4/6 (middle), and CDK2 (right) activity over time. Each row represents a single-cell trace. WM983B cells were treated with Vem (1 mM) alone (C) or combined with Cobi (100 nM) (D). Arrows and black dotted lines indicate the time of drug treatment or mitogen stimulation. (E) Mean ERK (left), CDK4/6 (middle), and CDK2 (right) activity traces in non-persister (gray) and persister (red) WM983B cells. Data represent mean ± 95% CI (non-persisters, n = 2,318 cells; persisters, n = 1,182 cells). (F) Mean ERK activity integrated for 5 h after mitogen stimulation in non-persister (gray) and persister (red) WM858 (left) and WM983B (right) cells. Data represent mean ± SD (n = 3 biological replicates). p values were calculated using two-tailed paired t tests (*p % 0.05). (G) Experimental design to assess the contribution of preexisting drug-resistance mutations to persister development. (H) Percentage of WM983B cells with geminin-degron signal. Sorted persisters and non-persisters were treated with a combination of Vem (1 mM) and Cobi (100 nM) after a 3-day drug holiday. As a control, drug-naive cells were treated with either DMSO or Vem/Cobi (n > 1,000 cells for each condition). See also Figure S3.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trypsin-EDTA, 0.25% 1X, phenol red, without Calcium and Magnesium Genesee Cat# 25-510 Bovine Serum Albumin (BSA) Protease-free powder Millipore Sigma Cat# A3311 5-Ethynyl-2’-deoxyuridine (EdU) Millipore Sigma Cat# 900584 AFDye 647 Picolyl Azide Click Chemistry Tools Cat# 1300 (+)-SODIUM L-ASCORBATE Millipore Sigma Cat# A4034 COPPER(II) SULFATE (CuSO4) Millipore Sigma Cat# C1297 Halt protease inhibitor cocktail Thermo Scientific Cat# 1861279 PhosSTOP Millipore Sigma Cat# 4906845001 Critical commercial assays Proteome Profiler Human Phospho-RTK Array Kit R&D Systems Cat# ARY001B GeneJET Plasmid Miniprep Kit Thermo Scientific Cat# K0503 NucleoBond Xtra Midi prep kit Macherey-Nagel Cat# 740410.100 Experimental models:
Techniques: Activation Assay, Translocation Assay, Activity Assay, Two Tailed Test, Control
Journal: Cell reports
Article Title: Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.
doi: 10.1016/j.celrep.2023.112570
Figure Lengend Snippet: Figure 3. Alterations in ERK signaling dynamics are associated with BRAFi resistance (A and B) Mean ERK activity traces in WM858 (A) and WM983B (B) cells over 6 days. Cells were treated with Vem (1 mM) 24 h before imaging and treatment was maintained throughout time-lapse acquisition. Data represent mean ± 95% CI (A, n = 300 cells; B, n = 339 cells). (C and D) Heatmaps of single-cell ERK activity traces over time in WM858 (C) and WM983B (D) cells. Drug-naive cells were treated with Vem (1 mM) 24 h before time-lapse imaging (non-resist). (E and F) Normalized ERK activity in WM858 (E) and WM983B (F) as a function of time since maximum ERK activation after mitogen stimulation. ERK activity is normalized by its minimum and maximum values. Solid line represents best fit. (G) Immunoblotting for phosphorylated and total ERK in drug-naive (left), non-resistant (middle), and Vem-resistant (right) cells. WM858 (top) and WM983B (bottom) cells were collected at the indicated times after mitogen stimulation. (H) Mean ERK (left), CDK4/6 (middle), and CDK2 (right) activity traces in drug-naive (blue), non-resistant (gray), and Vem-resistant (red) cells over time. Data represent mean ± 95% CI (n > 19,000 cells for each condition). See also Figure S4.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trypsin-EDTA, 0.25% 1X, phenol red, without Calcium and Magnesium Genesee Cat# 25-510 Bovine Serum Albumin (BSA) Protease-free powder Millipore Sigma Cat# A3311 5-Ethynyl-2’-deoxyuridine (EdU) Millipore Sigma Cat# 900584 AFDye 647 Picolyl Azide Click Chemistry Tools Cat# 1300 (+)-SODIUM L-ASCORBATE Millipore Sigma Cat# A4034 COPPER(II) SULFATE (CuSO4) Millipore Sigma Cat# C1297 Halt protease inhibitor cocktail Thermo Scientific Cat# 1861279 PhosSTOP Millipore Sigma Cat# 4906845001 Critical commercial assays Proteome Profiler Human Phospho-RTK Array Kit R&D Systems Cat# ARY001B GeneJET Plasmid Miniprep Kit Thermo Scientific Cat# K0503 NucleoBond Xtra Midi prep kit Macherey-Nagel Cat# 740410.100 Experimental models:
Techniques: Activity Assay, Imaging, Activation Assay, Western Blot
Journal: Cell reports
Article Title: Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.
doi: 10.1016/j.celrep.2023.112570
Figure Lengend Snippet: Figure 6. Targeting RTK signaling inhibits the development of persisters (A) Mean ERK activity after activation of opto-RTK in WM858 (left) and WM983B (right) cells. Cells were treated with Vem (1 mM)/Cobi (100 nM) together with DMSO, SOSi (RMC-0331, 1 mM; BI-3406, 1 mM), SHP2i (RMC-4550, 1 mM), or ERKi (SCH772984, 1 mM) 24 h before time-lapse imaging. Data represent mean ± 95% CI (n > 150 cells for each condition). (B) Mean ERK activity integrated for 2 h after opto-RTK activation in WM858 (left) and WM983B (right) cells. Data represent mean ± SD (n = 3 biological replicates). p values were calculated using one-way ANOVA (*p % 0.05, **p % 0.001, ***p % 0.0001). (C) Immunoblotting for p-AKT at an activating site on AKT1 (S473), t-AKT, p-ERK, t-ERK, and GAPDH in cells treated with Vem (1 mM)/Cobi (100 nM) plus the indicated drug for 24 h before light stimulation for 1 h. (D) Mean p-AKT/t-AKT ratio. Values were normalized to the condition without light stimulation. Data represent mean ± SD (n = 3 biological replicates). p values were calculated using one-way ANOVA (*p % 0.05, ***p % 0.0001). (E) Density scatterplots of Hoechst versus EdU signals, with cell density color coded in gray scale. WM983B cells expressing opto-RTK were treated with Vem (1 mM)/Cobi (100 nM) plus the indicated drug 3 days before light stimulation for 6 h and fixed 24 h after the start of opto-RTK activation (n = 2,000 cells for each condition). (F) Percentage of S-phase cells. Data represent mean ± SEM (n = 3 biological replicates). p values were calculated using one-way ANOVA (*p % 0.05, **p % 0.001). See also Figure S10.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trypsin-EDTA, 0.25% 1X, phenol red, without Calcium and Magnesium Genesee Cat# 25-510 Bovine Serum Albumin (BSA) Protease-free powder Millipore Sigma Cat# A3311 5-Ethynyl-2’-deoxyuridine (EdU) Millipore Sigma Cat# 900584 AFDye 647 Picolyl Azide Click Chemistry Tools Cat# 1300 (+)-SODIUM L-ASCORBATE Millipore Sigma Cat# A4034 COPPER(II) SULFATE (CuSO4) Millipore Sigma Cat# C1297 Halt protease inhibitor cocktail Thermo Scientific Cat# 1861279 PhosSTOP Millipore Sigma Cat# 4906845001 Critical commercial assays Proteome Profiler Human Phospho-RTK Array Kit R&D Systems Cat# ARY001B GeneJET Plasmid Miniprep Kit Thermo Scientific Cat# K0503 NucleoBond Xtra Midi prep kit Macherey-Nagel Cat# 740410.100 Experimental models:
Techniques: Activity Assay, Activation Assay, Imaging, Western Blot, Expressing
Journal: Cell reports
Article Title: Kinetics of RTK activation determine ERK reactivation and resistance to dual BRAF/MEK inhibition in melanoma.
doi: 10.1016/j.celrep.2023.112570
Figure Lengend Snippet: Figure 7. Suppression of RTK signaling reduces BRAFi/MEKi resistance persistence (A) Human RTK phosphorylation arrays in drug-naive (left) and Vem/Cobi-resistant (right) WM858 and WM983B cells. (B) Mean ERK activity trace in Vem/Cobi-resistant WM858 (left) and WM983B (right). Drug-resistant cells were treated with ErbB1i (erlotinib; 1 mM), ErbB1-3i (sapitinib; 1 mM), SHP2i (RMC-4550; 1 mM), or ERKi (SCH772984; 1 mM) 24 h before time-lapse imaging. Data represent mean ± 95% CI (n > 900 cells for each condition). (C) Mean ERK activity integrated for 5 h after mitogen stimulation in Vem/Cobi-resistant cells. Data represent mean ± SD (n = 3 biological replicates). p values were calculated using one-way ANOVA (*p % 0.05, ***p % 0.0001). (D) Clonogenic assay. Vem/Cobi-resistant cells were treated with the indicated drugs and stimulated with mitogens every 2–3 days for 28 days and stained with crystal violet. (E–G) Tumor growth curves (E and F) and tumor mass boxplot (G) (control, n = 7 mice; BRAFi, n = 7 mice; SHP2i, n = 7 mice; BRAFi + SHP2i, n = 8 mice). In the boxplot (G), the middle lines represent the median, boxes indicate the 25th and 75th percentiles, and whiskers denote the total range for each population. Data represent mean ± SEM (F). p values were calculated using one-way ANOVA (*p % 0.05, **p % 0.001, ***p % 0.0001). See also Figure S10.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Trypsin-EDTA, 0.25% 1X, phenol red, without Calcium and Magnesium Genesee Cat# 25-510 Bovine Serum Albumin (BSA) Protease-free powder Millipore Sigma Cat# A3311 5-Ethynyl-2’-deoxyuridine (EdU) Millipore Sigma Cat# 900584 AFDye 647 Picolyl Azide Click Chemistry Tools Cat# 1300 (+)-SODIUM L-ASCORBATE Millipore Sigma Cat# A4034 COPPER(II) SULFATE (CuSO4) Millipore Sigma Cat# C1297 Halt protease inhibitor cocktail Thermo Scientific Cat# 1861279 PhosSTOP Millipore Sigma Cat# 4906845001 Critical commercial assays Proteome Profiler Human Phospho-RTK Array Kit R&D Systems Cat# ARY001B GeneJET Plasmid Miniprep Kit Thermo Scientific Cat# K0503 NucleoBond Xtra Midi prep kit Macherey-Nagel Cat# 740410.100 Experimental models:
Techniques: Phospho-proteomics, Activity Assay, Imaging, Clonogenic Assay, Staining, Control