Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Scimp is required for sustained LPS responses and Scimp TV1 is basally, rather than LPS‐inducibly, phosphorylated. (a, b) BMMs from wild‐type (WT) and Scimp TV1 mice were treated with LPS (1 ng mL −1 ) for the indicated time points, before being washed of LPS and replated in fresh media for a further 24 h. Supernatants were collected and assessed for IL‐12p40 production by ELISA. Data (mean ± s.e.m., n = 3 or 4 mice per genotype) are combined from three or four independent experiments. (c) BMMs from WT and Scimp TV1 (TV1) mice were differentiated with either CSF‐1 (10 ng mL −1 ; lanes at left) or GM‐CSF (10 ng mL −1 ; lanes at right) for 6 days. CSF‐1‐BMMs were stimulated with LPS (10 ng mL −1 ) and IFNγ (5 ng mL −1 ) or GM‐CSF (10 ng mL −1 ) for 24 h. Total protein was collected and assessed for Scimp expression via western blot. Data are representative of two independent experiments collected from two mice per genotype. (d) BMMs from WT and Scimp TV1 mice were differentiated using CSF‐1, then primed overnight with GM‐CSF (10 ng mL −1 ). BMMs were stimulated with LPS (10 ng mL −1 ) for the indicated time points before being lysed. Collected protein was immunoprecipitated using a rabbit anti‐Scimp antibody or an isotype control (rabbit anti‐Myc tag). Immunoprecipitated samples were assessed for levels of phosphorylated Scimp (p‐Tyr) and coimmunoprecipitated TLR4 and Lyn via western blot. Data are representative of two independent experiments. (e) TLR4 and (f) phospho‐Scimp levels from d were quantified using Image Lab and displayed relative to the corresponding immunoprecipitated Scimp band (the IC background signal in control lanes was subtracted from the appropriate sample band intensity before quantifying). Data are representative of two independent experiments collected from two mice per genotype. (g) SH2 pulldown assays were used to assess LPS‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y120 (SLP‐SH2) on Scimp‐V5 in RAW264.7 cells expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments. Statistical significance in a and b was determined using two‐way ANOVA, followed by Bonferroni’s multiple comparison test using GraphPad Prism 9 (*** P < 0.001). GM‐CSF‐BMM, granulocyte macrophage colony‐stimulating factor‐derived bone marrow‐derived macrophages; IC, isotype control; IFN, interferon; LPS, lipopolysaccharide.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Immunoprecipitation, Derivative Assay

Journal: Immunology and Cell Biology

Article Title: An alternative downstream translation start site in the non‐TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages

doi: 10.1111/imcb.12540

Figure Lengend Snippet: Assessment of Scimp cellular localization and phosphorylation in response to CpG DNA. (a–c) Wild‐type (WT) BMMs retrovirally transduced with either an empty vector, Scimp‐V5 or Scimp TV1‐V5 expression construct. Cells were treated with 0.3 µM fluorescently labeled (Alexa‐647‐conjugated) CpG DNA (CpG) for 30 min. Cellular localization of V5‐tagged Scimp proteins and CpG DNA were assessed via immunofluorescence microscopy (anti‐V5/Scimp: green; CpG‐647: red; DAPI: blue; scale bars: 10 µm). White arrows indicate filopodia‐localized Scimp‐V5. Similar results were observed in three independent experiments in which a total of three WT C57Bl/6 mice were retrovirally transduced (1 mouse per experiment). (b) SH2 pulldown assays were used to assess CpG DNA‐inducible phosphorylation of Y58 (Grb2‐SH2) and Y96 (CSK‐SH2) on Scimp‐V5 in CSF‐1‐BMMs expressing Scimp‐V5 or Scimp TV1‐V5. Data are representative of three independent experiments and three mice per genotype. (c) CSK‐bound Scimp and Scimp TV1 bands from b were quantified and normalized to the 0‐min control of the WT. Data (mean ± s.e.m., n = 3 mice per genotype) are combined from three independent experiments. BMM, bone marrow‐derived macrophages.

Article Snippet: Lysates were passed through a 28‐gauge needle and centrifuged at 10 000 g for 15 min. 30 µL of sample was added to columns that contained 200 µg (20 µL of 10 µg per µL stock solution) of SH2 probe [SH2 domain of either human CSK, GRB2 or SLP65 conjugated to GST and bound to Pierce protein G plus agarose beads (Life Technologies) as described in Luo et al. ].

Techniques: Transduction, Plasmid Preparation, Expressing, Construct, Labeling, Immunofluorescence, Microscopy, Derivative Assay

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the healthy population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT and CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the HT population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: Biomarker Insights

Article Title: Comparative Analysis of Biomarkers in Type 2 Diabetes Patients With and Without Comorbidities: Insights Into the Role of Hypertension and Cardiovascular Disease

doi: 10.1177/11772719231222111

Figure Lengend Snippet: Important features in the CVD population with the use of LDA. Abbreviations: C5a, complement component 5a; 8-OHdG, deoxyguanosine; GSH, glutathione; GSSG, oxidized glutathione; HbA1c, hemoglobin A1C; HDL, high-density lipoprotein; IL-6, interleukin-6; IL-1β, interleukin-1β; IL-10, interleukin-10; LDL, high-density lipoprotein; MCP-1, monocyte chemoattractant protein-1; MOTSc, mitochondrial protein; p66Shc, adaptor protein; TC, total cholesterol.

Article Snippet: About 450 nm is absorbance.The Human SHC-Transforming Protein 1 ELISA kit (CUSA-BIO; Flarebio Biotech LLC) was used to measure p66shc because, according to the kit’s creators, it primarily detects Shc1 and its isoform p66shc.

Techniques:

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 through GHR signaling (A) Western blots of hNCC line #1 pretreated with 20 mg/mL pegvisomant (Peg) for 1 h then treated with GH (500 ng/mL) for an additional 24 h. C, untreated control. (B) Colon tissue derived from 3- and 24-month-old WT and GHR −/− male mice. Each lane represents sample analysis derived from an individual animal. (C) hNCC line #1 transduced with lentivirus expressing GH (Lenti-GH) or empty vector (Lenti-V) and analyzed 10 days later. (D) iPSC-derived human intestinal organoid lines from 3 different patients were transduced with Lenti-V or Lenti-GH and analyzed 5 weeks later. Representative blots from one human intestinal organoid line are shown. (E) hNCC line #1 transduced with lentivirus expressing control shRNA (shControl) or GH shRNA (shGH). (F) Three different human intestinal organoid lines were transduced with GFP-expressing Lenti-V or Lenti-GH and cultured for 5 weeks, then sorted for GFP-negative cells. GFP-negative cells co-existing in organoids with either Lenti-GH or Lenti-V expressing cells were analyzed. All lines exhibited similar results. Representative blots from one human intestinal organoid line are shown. Representative blots from at least 3 independent experiments are depicted. ImageJ quantifications of western blots are depicted in Figure S2 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Derivative Assay, Transduction, Expressing, Plasmid Preparation, shRNA, Cell Culture

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet: GH induces WIP1 by activating Src/AMPK (A and B) Western blots of (A) hNCC line #1 treated with 1 μM AMPK inhibitor (Compound C) for 1 h followed by GH (500 ng/mL) for an additional 24 h and (B) human intestinal organoids treated with 2 μM AMPK inhibitor and GH (500 ng/mL) for 48 h. (C–F) Western blots of hNCC line #1 was treated with (C) GH (500 ng/mL) for up to 60 min; (D) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 30 min–60 min; (E) 25 μM Src inhibitor for 1 h followed by GH (500 ng/mL) for 24 h; and (F) 25 μM Src inhibitor for 24 h followed by GH (500 ng/mL) for 3 h. Untreated cells/organoids served as control (C). (G–I) Western blots of hNCC line #1 transduced with lentivirus expressing Control shRNA (shControl) or Src shRNA (shSrc) (G) analyzed 72 h after transduction; (H) treated with GH (500 ng/mL) for 3 h; and (I) treated with GH (500 ng/mL) for 24 h. ImageJ quantifications of western blots are depicted in Figure S5 .

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Western Blot, Control, Transduction, Expressing, shRNA

Journal: iScience

Article Title: WIP1 is a novel specific target for growth hormone action

doi: 10.1016/j.isci.2023.108117

Figure Lengend Snippet:

Article Snippet: Src shRNA (human) lentiviral particles , Santa Cruz Biotechnology , Cat# sc-29228-V.

Techniques: Virus, Control, shRNA, Recombinant, Cell Recovery, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, DC Protein Assay, Extraction, Immunoprecipitation, cDNA Synthesis, Single Cell Gel Electrophoresis, Derivative Assay, Generated, Software, Imaging, Real-time Polymerase Chain Reaction, Microscopy

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 , 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 , 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: Signal Transduction and Targeted Therapy

Article Title: Membranous NOX5-derived ROS oxidizes and activates local Src to promote malignancy of tumor cells

doi: 10.1038/s41392-020-0193-z

Figure Lengend Snippet: NOX5 positively regulates Src activity. a Stable overexpression of NOX5 in the indicated ESCC cell lines tested by immunoblotting. GAPDH was used as a loading control. b Total protein lysates from the vector control or NOX5-overexpressing KYSE30 (upper panel) or KYSE410 (lower panel) cells were analyzed using antibody array against 43 kinase phosphorylation sites. c Silencing NOX5 in two specific short hairpin (sh) RNA-transduced stable ESCC cell lines examined by immunoblotting. GAPDH was used as a loading control. d vector control or NOX5-overexpressing KYSE30 (right panel) or KYSE410 (left panel) cells were pretreated with 10 μM NADPH oxidase inhibitor-DPI or control solvent for 90 min, respectively, or shRNA vector or NOX5 shRNA-1, shRNA-2, were cultured under normoxic and hypoxic conditions for 24 h. The Src activity was assayed by Src activation quantitative ELISA assay. e vector control or NOX5-overexpressing KYSE30 or KYSE410 cells were treated with ROS scavenger NAC (2 mM, pretreated with 90 min) or H 2 O 2 scavenger-PEG-catalase (400 units/ml) or control solvent, respectively, were cultured under normoxic and hypoxic conditions for 24 h. Src activity was assessed using quantitative ELISA assay. *** P < 0.001; two-tailed unpaired Student’s t -test. Error bars represent mean ± SD of five independent experiments. f NOX5 expression was associated with pSrc (Tyr 419 ), expression in 92 primary human ESCC specimens (cohort I). Two representative specimens with low and high levels of NOX5 were shown. Magnification, ×10 as indicated. Percentages of specimens showing low or high NOX5 expression relative to the level of pSrc. Statistical differences were evaluated using the chi-square test

Article Snippet: Levels of activated Src and Pyk2 in the cell lysis (~20 μg protein per sample) were measured using the human phosphor-Src (Tyr 419 ) ELISA kit (Raybiotech; catalog# PEL-SRC-Y419-T) and the human phosphor-Pyk2 (Tyr 402 ) ELISA kit (Raybiotech; catalog# PEL-PYK2-Y402-T) following the manufacturer’s protocols.

Techniques: Activity Assay, Over Expression, Western Blot, Plasmid Preparation, Ab Array, shRNA, Cell Culture, Activation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Expressing