phospho src y419 egfr rabbit polyclonal antibody  (R&D Systems)

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , b , Unsupervised hierarchical clustering of the phospho-catalytic activity signatures of WiDr cells treated with vemurafenib (VEM; n = 13 independent experiments) ± gefitinib (GEF; n = 5 independent experiments) or cetuximab (CET; n = 5 independent experiments) as compared to their untreated control counterparts ( n = 23 independent experiments). a , ATP consumption in cell extracts using 228 peptide sensors. b , Kinase signatures deconvoluted from the peptide phosphorylation profiles in a . Bar graphs next to the heatmaps show the P values (two-sided Student’s t test) for each of the peptides ( a ) or kinases ( b ) comparing all treated samples to controls. c , Volcano plot of the data in b displaying the change in kinase activity versus P value for each treatment arm (same as b : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments). d , Bar graphs of the data in b representing the shift in activity of SRC, SFK, EGFR and HER family kinases when cells were treated with vemurafenib alone or in combination with gefitinib or cetuximab. Kinase activity is compared to that in untreated control cells, and data are displayed as the average ± standard error in nM of ATP. Same as in b , c : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments. e , Representative IHC images showing staining intensity for active SFK (phosphorylated Y419 epitope in the SRC activation site) following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 d. The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition and n = 20 independent tissue areas per tumor and per condition. f , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ). Using batch processing and automated analysis of IHC images, protein expression was measured at the single-cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). g , Proposed parallel mechanism of SRC activation in response to BRAF/MEK/EGFR therapies in BRAF V600E CRC. BRAF*, BRAF V600E .

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activity Assay, Control, Phospho-proteomics, Staining, Activation Assay, Immunohistochemistry, Expressing

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines were treated with vemurafenib (VEM) for 7 to 8 hours. Vemurafenib was used at 1.75 uM in HT29, 2 uM in KM20, 0.15 uM in LIM2405, 2.25 uM in SNUC5, and 1.5 uM in WiDr (details of treatment conditions (concentration and time) are available in spreadsheets Supplementary Table and of the Supplementary Tables document). Cell lysates were assayed by western blot with the indicated antibodies. Upper panels: SFK activation is reflected by increased phosphorylation of the SRC activation site, Y419 (pY419). HSP90 is used as loading control. Bottom panel: reduction in ERK 1/2 phosphorylation as control of BRAF inhibition. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated ≥3 times with similar results. b , Representative IHC images showing total SRC staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis (see Methods for details). A scale bar is provided (100 micrometers). As in main Fig. , IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition, and n = 20 independent tissue areas per tumor and per condition. c , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. As in main Fig. , we used batch processing and automated analysis of IHC images to quantify protein expression at the single cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). d , SRC staining score by IHC in untreated patient CRC tumor specimens with or without a BRAF V600E mutation, from primary (prim.) or metastatic (met.) sites.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Concentration Assay, Western Blot, Activation Assay, Phospho-proteomics, Control, Inhibition, Molecular Weight, Staining, Immunohistochemistry, Expressing, Mutagenesis

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Shift in vemurafenib (VEM) sensitivity (mean fold-change ± standard error) measured via cell viability assays and calculation of the combination index (CI score; top panel) upon treatment of BRAF V600E or KRAS mutated or MAP3K8 amplified CRC, or melanoma (MEL) cell lines with VEM together with: a SRC inhibitor, dasatinib (DAS), saracatinib (SAR) or bosutinib (BOS), or an EGFR inhibitor, gefitinib (GEF), for three days. Same methods as in Fig. (n = 3 independent experiments per cell line and per drug combination). b , BRAF V600E CRC cell lines were transfected with a siRNA against CSK, a negative regulator of SFKs, and siRNA Control. Cell lysates were assayed by western blot with the indicated antibodies, showing the effect of CSK depletion on SFK activation. Molecular weight/size markers are indicated on the right (kDa). Experiment repeated 3 independent times with similar results. c , Bar graphs representing fold-change (log scale) ± standard error for change in sensitivity to VEM with knockdown of CSK in 3-day cell viability assays. Top panel: combination index, Bliss model score; colors as in Fig. (n = 4 independent experiments).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Amplification, Transfection, Control, Western Blot, Activation Assay, Molecular Weight, Knockdown

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Cell viability assays evaluating WiDr cells treated with vemurafenib plus a panel of kinase inhibitors selected on the basis of the results in Fig. . The size of each bubble indicates the magnitude of the change in kinase activity induced by vemurafenib treatment (Fig. ), with color signifying increased (yellow) or decreased (blue) activity; ( y axis, log 2 scale). r s is the Spearman’s rho correlation, and the P value is from a two-tailed t test. Drug combinations were tested in n = 89 independent experiments. b , Shift in vemurafenib sensitivity, measured by cell viability assay (left) and calculation of the CI (right), upon treatment of BRAF V600E CRC or melanoma (MEL) cell lines with vemurafenib together with a SRC inhibitor (dasatinib (DAS), saracatinib (SAR) or bosutinib (BOS)) or an EGFR inhibitor (gefitinib) for 3 d. CI scores are averaged from individual experimental CIs calculated at 1×GI 50 , 2×GI 50 and 0.5×GI 50 concentrations of each drug ( n ≥ 3 independent experiments). c , Colony formation assays in which BRAF V600E CRC or melanoma (Mel888) cells were treated with an increasing concentration of vemurafenib alone (control, CON) or with a fixed dose of dasatinib. Data are representative of n = 2 independent repeats. d , Western blots showing knockdown of SRC in BRAF V600E CRC cell lines stably transfected with a control shRNA (shCON) or two different SRC-targeting shRNAs (shSRC). HSP90 serves as a loading control. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated three independent times with similar results. e , Bar graphs representing the fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib with knockdown of SRC in 3-day cell viability assays. Top, CI, Bliss model score; colors as in b ( n = 3 independent experiments per cell line). f , Colony formation in BRAF V600E CRC cells treated with an increasing concentration of vemurafenib with or without SRC knockdown. Data are representative of n = 2 independent repeats.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activity Assay, Two Tailed Test, Viability Assay, Concentration Assay, Control, Western Blot, Knockdown, Stable Transfection, Transfection, shRNA, Molecular Weight

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Levels of pY419, non-pY530, and total SFK measured by western blot in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) were quantitated. Data are normalized to control untreated per cell line and displayed as average ± standard deviation measured across HT29, KM20, LIM2405, WiDr (data available in spreadsheet ‘Fig. ’ of the Source Data document; n = 12 independent experiments). b , Shift in vemurafenib (VEM) sensitivity, measured via cell viability assays and calculation of the combination index (CI score; top panel) upon treatment of BRAF V600E CRC or melanoma (MEL) cell lines with VEM + gefitinib (GEF) ± dasatinib (DAS) and VEM + DAS ± GEF, for three days. The addition of DAS to VEM + GEF increases sensitivity to VEM to a greater extent than the addition of GEF to VEM + DAS, highlighting the contribution of SFK and supporting that SFK activation upon VEM treatment is EGFR-independent. Same methods as in Fig. and Extended Data Fig. (n = 4 independent experiments per cell line). c and d , Mouse weight as a surrogate for toxicity following treatment of BRAF V600E CRC cell line xenografts ( c ) (n = 14 mice per group), or patent-derived xenografts ( d ) (n = 16 mice per group), with vehicle control or the inhibitors listed. Data is displayed as the average weight in grams ± standard deviation. e , Tumor growth inhibition in BRAF V600E CRC PDX models following treatment with VEM ± GEF ± DAS or vehicle (control). Waterfall plots show the relative change in tumor volume: each bar represents one tumor; and the height of the bar compares the final volume at day 21 to the starting volume at day 1. Volume changes are capped at 5-fold of the starting volume (that is 500%). Average final tumor volumes per treatment group are indicated underneath the graph (black font). Student t-test, two-sided, p-values are indicated when p < 0.05. Tumors that regressed by day 21 compared to volume at mid-treatment (that is, day 10) are shown in purple; percentages of regressing tumors per group are indicated underneath the graph. f , The GLM p-values corrected for false discovery rate (FDR) corresponding to the main Fig. , are shown (n = 8 mice per treatment group per PDX model).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Western Blot, Control, Standard Deviation, Activation Assay, Derivative Assay, Inhibition

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib were lysed and immunoblotted with the indicated antibodies. SFK activation is reflected by increased phosphorylation of the SRC activation site Y419 (pY419) and lack of phosphorylation of the inhibitory site Y530 (non-pY530). Active SRC can be deactivated by rephosphorylation of Y530 by CSK. HSP90 serves as a loading control. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated three times with similar results. b , Shift in vemurafenib sensitivity measured by cell viability assay (left) and calculation of the CI (right) upon treatment of BRAF V600E CRC or melanoma cell lines with vemurafenib + gefitinib ± a SRC inhibitor, dasatinib, for 3 d ( n = 4 independent experiments per cell line). c , Colony formation assays in which BRAF V600E CRC cells were treated with an increasing concentration of vemurafenib alone (control) or with a fixed dose of gefitinib ± dasatinib. Data are representative of n = 2 independent repeats. d , Treatment of cell line-derived xenograft mouse models with a vemurafenib progenitor, PLX4720 (PLX); dasatinib; saracatinib; and/or gefitinib for 21 d ( n = 7 mice per group). Plotted is the percent change in tumor volume relative to baseline (day 1). Data are displayed as the average for all mice in a specified treatment group ± standard error. e , Treatment of PDX models with vemurafenib ± gefitinib ± dasatinib for 21 d, with data plotted as in d ( n = 8 mice per group). All raw and relative tumor volumes and exact P values shown in d , e are available as Source Data; P values are from a two-sided Student’s t test. f , g , GLMs testing the association of change in tumor volume between treatment arms and vehicle over time shown in d , e . Effect size is measured as the GLM standard coefficient. A GLM was applied to each tumor model separately or combined. Results for cell line xenografts and PDXs are shown in f and g , respectively. GLM P values corrected for FDR are shown in g . NT, not tested. h , i , Comparison of the effect sizes and FDR-corrected P values of treatment arms with and without a SRC inhibitor. The same number of mice per group shown in d , e was used for the analyses in f – i (that is, n = 7 mice per treatment group for WiDr and KM20 cell line xenografts and n = 8 mice per treatment group for PDX models 1 and 2).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activation Assay, Phospho-proteomics, Control, Molecular Weight, Viability Assay, Concentration Assay, Derivative Assay, Comparison

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Western blots to detect phospho-T202/Y204 ERK1/2 and total ERK1/2 in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) or dasatinib (DAS) collected after 8 h, 24 h, 48 h or 72 h. HSP90 is used as a loading control. The experiment was repeated 2 independent times with similar results. b , Quantification of western blots shown in panel ( a ). The bar plot (averages and standard deviations per treatment condition across cell lines) was overlaid with a dot plot displaying individual measurements per cell line and condition. Data are normalized to p-ERK levels after 8 h treatment with VEM alone. See table below for detailed values and color codes; n = 8 cell lines. c , Western blots to detect total and phospho-Y654 beta-catenin (CTNNB1) in BRAF V600E CRC cell lines treated with VEM, or GEF, or DAS, or combinations of VEM + GEF, or VEM + DAS, or VEM + GEF + DAS. The detection of phospho-Y419 and total SFK serves as a control for the effect of SFK-inhibition (with DAS). The experiment was repeated 3 independent times with similar results. In panels a , c , molecular weight/size markers are indicated on the right (kDa).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Western Blot, Control, Inhibition, Molecular Weight

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Western blots of BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib or dasatinib. The Y654 residue of CTNNB1 is a reported phospho-target site of SRC kinases . ERK1/ERK2 phospho-T202/Y204 serves as a control for the effect of BRAF inhibition (with vemurafenib). SFK pY419 serves as a control for the effect of SFK inhibition (with dasatinib). Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated three independent times with similar results. b , Color-coded expression levels of β-catenin target genes ( MYC , AXIN2 , ASCL2 , S100A6 , LEF1 , NOTCH2 , SP5 ) measured using qRT–PCR in BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib or dasatinib. Expression profiles are shown as fold change against the mean mRNA expression level with vemurafenib, vemurafenib + gefitinib, vemurafenib + dasatinib. Percentages indicate the proportion of measurements across eight cell lines where the expression of the indicated gene (top) was lower with vemurafenib + dasatinib than with vemurafenib + gefitinib or vemurafenib alone. The right-most columns indicate P values (Student’s t test) comparing gene expression for vemurafenib + dasatinib versus vemurafenib alone. NA, not available due to expression levels that were too low. n ≥ 3 independent experiments. c , The expression profiles in b averaged across all eight cell lines ( n = 4 independent experiments). Exact P values for b , c are available as Source Data. d , Proposed mechanism regulated by SRC that drives resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Western Blot, Residue, Control, Inhibition, Molecular Weight, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Levels of secreted PGE2 were measured by ELISA in the conditioned medium of BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib. Data are displayed as the average PGE2 secretion in pg ml –1 per 100,000 cells ± s.d. ( n = 3 independent experiments per cell line). b , BRAF V600E CRC cell lines were treated with exogenous PGE2. Cell lysates were assayed by western blot as indicated. Y419 phosphorylation and lack of phosphorylation of Y530 (non-pY530) are used as readouts of SFK activation. HSP90 serves as a loading control. The experiment was repeated two independent times per cell line with similar results. c , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib upon further treatment with PGE2 or untreated control in 3-day cell viability assays. Top, CI, Bliss model. Same methods as in Fig. ( n = 3 independent experiments per cell line). d , Western blots to detect pY654 of CTNNB1 in BRAF V600E CRC cell lines treated with exogenous PGE2. The experiment was repeated two independent times per cell line with similar results. e , Three BRAF V600E CRC cell lines engineered with a doxycycline-inducible constitutively active GNAS construct, iGNAS R201C , were treated with doxycycline. Cell lysates were assayed by western blot as indicated. The experiment was repeated three times with similar results. f , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib after iGNAS R201C induction in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). g , GNAS was knocked out in BRAF V600E CRC cells using CRISPR (GNAS-KO). GNAS knockout was validated by western blot (top). GNAS-KO cells were treated with vemurafenib, and cell lysates were assayed by western blot with the indicated antibodies (bottom). The experiment was repeated ≥2 times with similar results. In b , d , e , g , molecular weight/size markers are indicated on the right (kDa). h , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib with GNAS knockout in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). i , Representative IHC images showing COX2 staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib and/or trametinib for 3 or 21 d (where n is the same as defined in Fig. ). The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). j , Quantification of COX2 staining intensity by IHC for two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ; n is the same as defined in Fig. ). k , Proposed mechanism of COX2–PGE2-mediated SRC-driven resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Activation Assay, Control, Construct, CRISPR, Knock-Out, Molecular Weight, Staining

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , b , Tumor growth profiles in BRAF V600E CRC PDX models 1 and 2 treated with encorafenib (ENC) ± panitumumab ± celecoxib or vehicle (VEH; control). Changes in tumor volume relative to starting volume at day 1 (average ± standard error) are plotted over time. P values from two-sided Student’s t tests across all time points comparing treatment arms are shown as a grayscale underneath each graph. NS, not significant; X, not available. All raw and relative tumor volumes are available as Source Data. In a , n = 11 mice per treatment group; in b : VEH, n = 5 mice; ENC, n = 6 mice; ENC + PAN, n = 9 mice; ENC + PAN + CEL, n = 9 mice. c , GLMs to test the association of change in tumor volume over time, either between treatment arms and vehicle (left) or between combination therapy and encorafenib alone (right). A GLM was applied to each individual PDX model and to both PDXs combined. Top, effect size measured as the GLM standard coefficient comparing the efficacy of the treatment arms. Bottom, GLM FDR-corrected P values. d , Comparison of effect sizes and FDR-corrected P values of treatment arms with and without celecoxib, using vehicle or encorafenib treatment as the baseline (left and right, respectively). The same number of mice per group shown in a , b was used for the analyses in c , d . e , Mouse weight as a surrogate for treatment toxicity. Data are displayed as the average weight in grams ± s.d. All weights from the results shown in a , b were used: VEH, n = 16 mice; ENC, n = 17 mice; ENC + PAN, n = 20 mice; ENC + PAN + CEL, n = 20 mice. f , Schematic summary of the states of signaling pathways depending on treatment: (1) untreated tumors, with BRAF–MEK–ERK as the main driver of progression (red) and baseline activity of the EGFR and COX2–SRC signaling pathways (gray), and (2–4) tumors treated with drugs (listed on top) inhibiting the activity (blue) of the three distinct signaling axes: BRAF–MEK, EGFR and COX2–SRC–β-catenin In scenario (4), triple treatment collectively blocks the cooperative dependencies that drive resistance and progression.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Control, Comparison, Protein-Protein interactions, Activity Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: The short-chain fatty acid butyrate exerts a specific effect on VE-cadherin phosphorylation and alters the integrity of aortic endothelial cells

doi: 10.3389/fcell.2023.1076250

Figure Lengend Snippet: BUT engages Src kinases for specific tyrosine phosphorylation at VEC. (A) HAOECs were treated for 15 min before and during BUT incubation with PP2 (10 μM) or vehicle (DMSO). Subsequently, phospho-VECY731 levels were analyzed by western blotting. Phospho-signals of n = 4 experiments were quantified by densitometry. (B) Immune fluorescent microscopy of HAOECs treated with the vehicle (DMSO) or PP2 and BUT (5 mM) for 1 h and subsequently stained for VEC and general phospho-tyrosine residues. Scale bar (10 μm). (C) Validation of Src KD by RNAi. HAOECs were transfected with Src-specific siRNA or control-siRNA. Cells were lysed and analyzed for Src expression 12 h later by western blotting. (D) Stimulation of Src KD and control cells with BUT (1 mM) for 1 h and analysis of phosho-VECY731 expression as shown in . Quantification corresponds to n = 6 experiments. (E) Phospho-SrcY416 western blotting of BUT-treated or -untreated cells, transfected with Ctrl or FFAR3 siRNA. Data display densiometric quantification of relative phospho-Src signals normalized to the level in BUT-untreated Ctrl siRNA-transfected cells (dashed red line) of n = 3 experiments.

Article Snippet: We used the following commercially available antibodies for western blotting and immunofluorescence microscopy: monoclonal mouse anti-p-Tyr (PY99, Santa Cruz, sc-7020), polyclonal rabbit anti-phospho-VEC (against phospho-Tyr731, Invitrogen 44-1145G) , polyclonal rabbit anti-phospho-VEC (against phospho-Tyr685, Abcam, ab119785) , polyclonal rabbit anti-phospho-VEC (against phospho-Tyr658, Invitrogen, 44-1144G) , monoclonal mouse anti-human VEC (clone F-8, Santa Cruz, sc-9989), monoclonal mouse anti-human VEC (BV6, Millipore, MABT134), monoclonal mouse anti-human SRC (Invitrogen, AHO1152), monoclonal rabbit anti-human phospho-SRC (against phospho-Tyr 416 (D49G4), Cell Signaling, 6943), monoclonal rabbit anti-human VEC (E6N7A, Cell Signaling, 93467S), and monoclonal mouse anti-human ß-actin (Cell Signaling, 3700).

Techniques: Incubation, Western Blot, Microscopy, Staining, Transfection, Expressing

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a , b , Unsupervised hierarchical clustering of the phospho-catalytic activity signatures of WiDr cells treated with vemurafenib (VEM; n=13) ± gefitinib (GEF; n=5) or cetuximab (CET; n=5) as compared to their untreated control counterparts (n=23) . a , ATP consumption in cell extracts using 228 peptide sensors. b , Kinase signatures deconvoluted from peptide phosphorylation profiles in ( a). Bar graphs next to the heatmaps display the p-values (p-val) for each of the peptides ( a ) or kinases ( b ) comparing all treated samples to controls. c , Volcano plot of data in ( b ) displays change in kinase activity versus p-values for each treatment arm. d , Bar graphs representing the shift in activity of SRC, SFK, EGFR and HER family kinases when cells are treated with VEM alone or in combination with GEF or CET. Kinase activity is compared to the untreated control cells and data displayed as the average ± standard error. e , Representative IHC images showing active SFK (phosphotyrosine-419 epitope in the SRC activation site) staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis. f , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. g, Proposed parallel mechanism of SRC activation in response to BRAF/MEK/EGFR therapies in BRAF V600E CRC. BRAF*: BRAF V600E .

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Activity Assay, Control, Phospho-proteomics, Activation Assay, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a, BRAF V600E CRC cell lines were treated with vemurafenib (VEM) for 7 to 8 hours. Vemurafenib was used at 1.75 uM in HT29, 2 uM in KM20, 0.15 uM in LIM2405, 2.25 uM in SNUC5, and 1.5 uM in WiDr (details of treatment conditions (concentration and time) are provided in the spreadsheet Suppl_Table S1 and Suppl_Table S2 in the supplementary XLS document Source Data & Experimental Conditions ). Cell lysates were assayed by western blot with the indicated antibodies. Upper panels: SFK activation is reflected by increased phosphorylation of the SRC activation site, Y419 (pY419). HSP90 is used as loading control. Bottom panel: reduction in ERK 1/2 phosphorylation as control of BRAF inhibition. b, Representative IHC images showing total SRC staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis. c, Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. d, SRC staining score by IHC in untreated patient CRC tumor specimens with or without a BRAF V600E mutation, from primary (prim.) or metastatic (met.) sites.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Concentration Assay, Western Blot, Activation Assay, Phospho-proteomics, Control, Inhibition, Staining, Immunohistochemistry, Mutagenesis

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a , Cell viability assays evaluating WiDr cells treated with vemurafenib (VEM) plus a panel of kinase inhibitors selected based on results in . The size of the bubble indicates the magnitude of the change in kinase activity induced by VEM treatment , with color signifying increased (yellow) or decreased (blue) activity; y-axis: log2 scale; r s : Spearman’s rho correlation, p: p-value for 2-tailed t-test. b , Shift in VEM sensitivity, measured via cell viability assays (left panel) and calculation of the combination index (right panel) upon treatment of BRAF V600E CRC or melanoma (MEL) cell lines with VEM together with a SRC inhibitor: dasatinib (DAS), saracatinib (SAR) or bosutinib (BOS), or an EGFR inhibitor: gefitinib (GEF), for three days. CI scores are averaged from individual experimental CIs calculated at 1x GI50, 2xGI50, 0.5xGI50 concentrations of each drug (n≥2). c , Colony formation assays where BRAF V600E CRC or melanoma (Mel888) cells were treated with an increasing concentration of VEM alone (control, CON) or with a fixed dose of DAS. d , Western blot showing knockdown of SRC in BRAF V600E CRC cell lines stably transfected with a control shRNA (shCON) or two different SRC-targeting shRNAs (shSRC). HSP90 serves as loading control. e , Bar graphs representing fold-change (log2 scale) ± standard error for change in sensitivity to VEM with knockdown of SRC in 3-day cell viability assays. Top panel: combination index, Bliss model score; colors as in , Colony formation in BRAF V600E CRC cells treated with an increasing concentration of VEM with or without SRC knockdown.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Activity Assay, Concentration Assay, Control, Western Blot, Knockdown, Stable Transfection, Transfection, shRNA

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a, Shift in vemurafenib (VEM) sensitivity, measured via cell viability assays and calculation of the combination index (CI score; top panel) upon treatment of BRAF V600E or KRAS mutated or MAP3K8 amplified CRC, or melanoma (MEL) cell lines with VEM together with: a SRC inhibitor, dasatinib (DAS), saracatinib (SAR) or bosutinib (BOS), or an EGFR inhibitor, gefitinib (GEF), for three days. b, BRAF V600E CRC cell lines were transfected with a siRNA against CSK, a negative regulator of SFKs, and siRNA Control. Cell lysates were assayed by western blot with the indicated antibodies, showing the effect of CSK depletion on SFK activation. c, Bar graphs representing fold-change (log scale) ± standard error for change in sensitivity to VEM with knockdown of CSK in 3-day cell viability assays. Top panel: combination index, Bliss model score; colors as in .

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Amplification, Transfection, Control, Western Blot, Activation Assay, Knockdown

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a, Levels of pY419, non-pY530, and total SFK measured by western blot in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) were quantitated. Data are normalized to control untreated per cell line and displayed as average ± standard deviation measured across HT29, KM20, LIM2405, WiDr (data provided in the spreadsheet ‘ ’ in the supplementary XLS document Source Data & Experimental Conditions ). b , BRAF V600E CRC cell lines were treated with exogenous PGE2. b, Shift in vemurafenib (VEM) sensitivity, measured via cell viability assays and calculation of the combination index (CI score; top panel) upon treatment of BRAF V600E CRC or melanoma (MEL) cell lines with VEM + gefitinib (GEF) ± dasatinib (DAS) and VEM + DAS ± GEF, for three days. The addition of DAS to VEM+GEF increases sensitivity to VEM to a greater extent than the addition of GEF to VEM+DAS, highlighting the contribution of SFK and supporting that SFK activation upon VEM treatment is EGFR-independent. c and d, Mouse weight as a surrogate for toxicity following treatment of BRAF V600E CRC cell line- ( c ) or patent-derived xenografts ( d ) with vehicle control or the inhibitors listed (PLX: PLX4720; SAR: saracatinib). Data is displayed as the average weight in grams ± standard deviation. e, Tumor growth inhibition in BRAF V600E CRC PDX models following treatment with VEM ± GEF ± DAS or vehicle (control). Waterfall plots show the relative change in tumor volume: each bar represents one tumor; and the height of the bar compares the final volume at day 21 to the starting volume at day 1. Volume changes are capped at 5-fold of the starting volume (i.e. 500%). Average final tumor volumes per treatment group are indicated underneath the graph (black font). T-test p-values are indicated when p<0.05. Tumors that regressed by day 21 compared to volume at mid-treatment (i.e., day 10) are shown in purple; percentages of regressing tumors per group are indicated underneath the graph. f, The GLM p-values corrected for false discovery rate (FDR) corresponding to the main , are shown.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Western Blot, Control, Standard Deviation, Activation Assay, Derivative Assay, Inhibition

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a , Western blot of BRAF V600E CRC cell lines treated with VEM ± GEF or DAS. The tyrosine 654 (Y654) of beta-catenin (CTNNB1) is a reported phospho-target site of SRC kinases . ERK1/2 phospho-T202/Y204 serves as a control for the effect of BRAF-inhibition (with VEM). SFK phospho-Y419 serves as a control for the effect of SFK-inhibition (with DAS). b , Color-coded expression levels of beta-catenin target genes ( MYC, AXIN2, ASCL2, S100A6, LEF1, NOTCH2, SP5 ) measured using quantitative real time (qRT) PCR in BRAF V600E CRC cell lines treated with VEM ± GEF or DAS. Expression profiles are shown as fold change against the mean mRNA expression level in VEM, VEM+GEF, VEM+DAS. Percentages indicate the proportion of measurements across 8 cell lines where the expression of the indicated gene (top) was lower with VEM+DAS than with VEM+GEF or VEM alone. The right-most columns indicate p-values (student t-test) comparing gene expression in VEM+DAS versus VEM alone. n.a.: not available due to expression levels that were too low. c , The expression profiles displayed in ( b ) were averaged across cell lines. d , Proposed mechanism regulated by SRC and that drives resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Western Blot, Control, Inhibition, Expressing, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a, Western blots to detect phospho-T202/Y204 ERK1/2 and total ERK1/2 in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) or dasatinib (DAS) collected after 8h, 24h, 48h or 72h. HSP90 is used as a loading control. b, Quantification of western blots shown in panel ( a ). The bar plot (averages and standard deviations per treatment condition across cell lines) was overlaid with a dot plot displaying individual measurements per cell line and condition. Data are normalized to p-ERK levels after 8h treatment with VEM alone. See table below for detailed values and color codes. c, Western blots to detect total and phospho-Y654 beta-catenin (CTNNB1) in BRAF V600E CRC cell lines treated with VEM, or GEF, or DAS, or combinations of VEM + GEF, or VEM + DAS, or VEM + GEF + DAS. The detection of phospho-Y419 and total SFK serves as a control for the effect of SFK-inhibition (with DAS).

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Western Blot, Control, Inhibition

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a , PGE2 secreted levels were measured by ELISA in the conditioned media of BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF). Data is displayed as the average PGE2 secretion in pg/mL per 100,000 cells ± standard deviation . b , BRAF V600E CRC cell lines were treated with exogenous PGE2. Cell lysates were assayed by western blot as indicated. Y419 phosphorylation and lack of phosphorylation of Y530 (non-pY530) are used as readouts of SFK activation. HSP90 serves as loading control. c, Bar graphs representing fold-change (log2 scale) ± standard error for change in sensitivity to VEM upon further treatment with PGE2 or untreated control (CON) in 3-day cell viability assays. Top panel: combination index, Bliss model score. d , Three BRAF V600E CRC cell lines engineered with a doxycycline-inducible constitutively active GNAS construct, iGNAS R201C , were treated with doxycycline. Cell lysates were assayed by western blot as indicated. e , Bar graphs representing fold-change (log2 scale) ± standard error for change in sensitivity to VEM or VEM+GEF after iGNAS R201C induction in 3-day cell viability assays. Top panel: combination index, as in , GNAS was knocked out in BRAF V600E CRC cells using CRISPR (GNAS-KO). GNAS-KO was validated by western blot (top panel). GNAS-KO cells were treated with VEM and cell lysates were assayed by western blot with the indicated antibodies (bottom panel). g, Bar graphs representing fold-change (log2 scale) ± standard error for change in sensitivity to VEM or VEM+GEF with GNAS-KO in 3-day cell viability assays. Top panel: combination index, as in , Representative immunohistochemistry (IHC) images showing COX2 staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis. i , Quantification of COX2 staining intensity by IHC for two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. j , Proposed mechanism of COX2/PEG2 mediated SRC-driven resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation, Western Blot, Phospho-proteomics, Activation Assay, Control, Construct, CRISPR, Immunohistochemistry, Staining

Journal: bioRxiv

Article Title: A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF V600E colorectal tumors

doi: 10.1101/2022.10.20.512885

Figure Lengend Snippet: a-b , Tumor growth profiles in BRAF V600E CRC PDX models #1 and #2 treated with ENC ± PAN ± CEL or vehicle (control). Changes in tumor volumes relative to starting volume at day 1 (average ± standard error) are plotted over time. Student t-test p-values across all time points comparing treatment arms are shown as a grey scale underneath each graph. n.s. : not significant; x : not available. All raw and relative tumor volumes can be found in the spreadsheets ‘ ’ and ‘ ’ of the Source Data & Experimental Conditions document. c , GLM to test the association of change in tumor volume over time, either between treatment arms and vehicle (left panel) or between combination therapy and ENC alone (right panel). GLM was applied to each individual PDX model and to both PDXs combined. The top section shows effect size measured as the GLM standard coefficient comparing the efficacy of the treatment arms. The bottom section shows GLM FDR-corrected p-values. d , Comparison of effect size and FDR-corrected p-values of treatment arms with and without celecoxib, using vehicle or ENC treatment as the baseline (respectively left and right). e , Mouse weight as a surrogate for treatment toxicity. Data is displayed as the average weight in grams ± standard deviation. f , Schematic summary of the states of signaling pathways depending on treatment: (i) untreated tumors, with BRAF-MEK-ERK as the main driver of progression (red), and baseline activity of the EGFR and COX2 / SRC signaling pathways (grey); (ii-iv) tumors treated with drugs (listed on top) inhibiting the activity (blue) of the three distinct signaling axes: BRAF-MEK, EGFR and COX2-SRC-beta Catenin; (iv) triple treatment collectively blocks the cooperative dependencies that drive resistance and progression.

Article Snippet: To detect phospho-Y419 SRC, phospho-Src (Y419) EGFR rabbit polyclonal supplied by R&D Systems (Cat# AF2685) was used at a titration of 1:50.

Techniques: Control, Comparison, Standard Deviation, Protein-Protein interactions, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Coimmunoprecipitation of CEACAM1, Src, LB1, and Annexin A2 with CD36. CD36 was immunoprecipitated from Ser508A mutant HepG2 cells, pretreated or not with insulin, run on SDS gels, and immunoblotted for CD36, CEACAM1, Src, LKB1, and Annexin A2 (AnxA2). Equal amounts of protein were probed on immunoblots.

Article Snippet: Blots were immunostained with goat anti-human CD36 (R&D, AF1955), mouse anti-human CEACAM1 (T84.1), rabbit anti-human Src (Abcam, AB109381), mouse anti-human LKB1 (Invitrogen, AH01392), mouse anti-human 4G10 (Sigma-Aldrich, 05-321), goat anti-human Annexin A2 (R&D, AF3928-SP), rabbit anti-human phospho-Src (R&D, MAB2685), rabbit β-Catenin (Abcam, Ab32572), rabbit anti-human PCSK9 (Invitrogen, AH01392), mouse anti-human AMPKα (Invitrogen, AH01332), or mouse anti-human FYN (Invitrogen, MA1-19331).

Techniques: Immunoprecipitation, Mutagenesis, Western Blot

Journal: Life Science Alliance

Article Title: Tyrosine phosphorylation of lamin A by Src promotes disassembly of nuclear lamina in interphase

doi: 10.26508/lsa.202101120

Figure Lengend Snippet: (A) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h and then lysed. Equal amounts of the whole cell lysates (WCLs) were incubated with anti-lamin A/C or pre-immune serum (IgG) as the control. The immunonocomplexes were analyzed by immunoblotting (IB) with anti-PY or anti-lamin A/C antibodies. An equal amount of WCLs was analyzed by immunoblotting with anti-Src, anti-Src pY416, or anti-actin. The tyrosine phosphorylation of lamin A was quantified and expressed as -fold relative to the level of MCF7 without dasatinib. (B) MCF7 and MDA-MB-231 cells were treated with (+) or without (−) 50 nM dasatinib for 1 h. The cells were fixed and stained for lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The nuclear circularity (4π × area/perimeter 2 ) was determined. The P -values were calculated from at least 150 cells pooled from three independent experiments. The percentage of the cells with nuclear lobulation was measured (n ≥ 400). The values (mean ± SD) are from three experiments. *** P < 0.001. (C) MDA-MB-231 cells were infected with lentivirues capable of expressing FLAG-lamin A or the mutants (Y45F and Y45D) and selected in the medium with neomycin (neo). An equal amount of WCLs was analyzed by immunoblotting (IB) with antibodies as indicated. (D) The cells as described in panel C were fixed and stained for FLAG-lamin A, lamin B1, and DNA. Representative images are shown. Scale bars, 10 μm. The percentage of the cells with nuclear lobulation was measured (n ≥ 900). Values (means ± SD) are from three experiments. * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are available for this figure.

Article Snippet: The rabbit polyclonal anti-Src pY416 (MAB2685) antibody was purchased from R&D Systems.

Techniques: Incubation, Control, Western Blot, Phospho-proteomics, Staining, Infection, Expressing