anti egfr  (Abcam)

 
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    Name:
    Anti EGFR phospho Y1092 antibody EP774Y
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    Catalog Number:
    ab40815
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    Structured Review

    Abcam anti egfr
    Expression of thymidylate synthase (TS) and related <t>EGFR</t> signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, <t>AKT,</t> and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    https://www.bioz.com/result/anti egfr/product/Abcam
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    anti egfr - by Bioz Stars, 2020-11
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    Images

    1) Product Images from "Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts"

    Article Title: Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts

    Journal: Thoracic Cancer

    doi: 10.1111/1759-7714.12818

    Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P
    Figure Legend Snippet: Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    Techniques Used: Expressing, Mouse Assay, End-sequence Profiling, Western Blot, Standard Deviation

    2) Product Images from "CD60b: Enriching Neural Stem/Progenitor Cells from Rat Development into Adulthood"

    Article Title: CD60b: Enriching Neural Stem/Progenitor Cells from Rat Development into Adulthood

    Journal: Stem Cells International

    doi: 10.1155/2017/5759490

    CD60b is expressed in different populations in the adult SVZ. Confocal images of adult SVZ sections showing the expression of CD60b (a, d, g, j, and m in red) and doublecortin (b-c), EGFR (e-f), GFAP (h-i), PNA (k-l), and vimentin (n-o) (in green). Nuclei were counterstained with TO-PRO-3 (blue). We observed the expression of CD60b in DCX (expressed in neuroblasts), R-EGF (expressed mostly in type C cells), GFAP (expressed in type B cells and astrocytes), and vimentin-positive cells (expressed in radial glia-like cells) (arrows). PNA, which was described as a negative marker of neural stem cells, and CD60b showed little colocalization (l). High magnification is showed in the insets. LV: lateral ventricle; DCX: doublecortin. Scale bar: 20 μ m.
    Figure Legend Snippet: CD60b is expressed in different populations in the adult SVZ. Confocal images of adult SVZ sections showing the expression of CD60b (a, d, g, j, and m in red) and doublecortin (b-c), EGFR (e-f), GFAP (h-i), PNA (k-l), and vimentin (n-o) (in green). Nuclei were counterstained with TO-PRO-3 (blue). We observed the expression of CD60b in DCX (expressed in neuroblasts), R-EGF (expressed mostly in type C cells), GFAP (expressed in type B cells and astrocytes), and vimentin-positive cells (expressed in radial glia-like cells) (arrows). PNA, which was described as a negative marker of neural stem cells, and CD60b showed little colocalization (l). High magnification is showed in the insets. LV: lateral ventricle; DCX: doublecortin. Scale bar: 20 μ m.

    Techniques Used: Expressing, Marker

    3) Product Images from "Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor"

    Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor

    Journal: Cell Proliferation

    doi: 10.1111/cpr.12858

    Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry
    Figure Legend Snippet: Generation, isolation, and characterization of epidermal growth factor receptor (EGFR)‐specific chimeric antigen receptor (CAR)‐engineered natural killer (NK) cells (EGFR‐CAR NK cells). (A) Flow cytometric analysis of phenotypic and subset composition of peripheral blood mononuclear cells (PBMCs) labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE. (B‐C) Flow cytometric analysis of phenotypic and subset composition of NK cells labeled with anti‐CD3‐PE‐Cy7, anti‐CD56‐PE, and anti‐CD69‐APC‐Cy7. (D) The percentage of CD3‐/CD56 + cells in day 0 PBMCs and day14 PBMC culture. (E) Real‐time PCR and (F) Western blotting analyses of the expression of exogenous CD3ζ in the non‐transduced NK cells, Con‐CAR NK cells, EGFR‐CAR‐1 NK cells, and EGFR‐CAR‐2 NK cells. β‐actin was used as an endogenous control. (G) The transduced NK cells stained with IgG‐FITC and EGFR‐FITC antibodies were detected by flow cytometry

    Techniques Used: Isolation, Labeling, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining, Flow Cytometry

    4) Product Images from "Andrographolide Suppresses Proliferation of Nasopharyngeal Carcinoma Cells via Attenuating NF- κB Pathway"

    Article Title: Andrographolide Suppresses Proliferation of Nasopharyngeal Carcinoma Cells via Attenuating NF- κB Pathway

    Journal: BioMed Research International

    doi: 10.1155/2015/735056

    Andro reduced the expression of NF- κ B target genes. (a) Andro induced downregulation of NF- κ B target genes EGFR, cyclin D1, and survivin. (b) Andro reduced expression of MMP-9, ICAM-1, and VEGF in NPC cell lines. Actin served as the loading control. Compared with control (DMSO) group, # P
    Figure Legend Snippet: Andro reduced the expression of NF- κ B target genes. (a) Andro induced downregulation of NF- κ B target genes EGFR, cyclin D1, and survivin. (b) Andro reduced expression of MMP-9, ICAM-1, and VEGF in NPC cell lines. Actin served as the loading control. Compared with control (DMSO) group, # P

    Techniques Used: Expressing

    5) Product Images from "Sofosbuvir Activates EGFR-Dependent Pathways in Hepatoma Cells with Implications for Liver-Related Pathological Processes"

    Article Title: Sofosbuvir Activates EGFR-Dependent Pathways in Hepatoma Cells with Implications for Liver-Related Pathological Processes

    Journal: Cells

    doi: 10.3390/cells9041003

    Sofosbuvir increases the expression and activation of EGFR. ( a , b ) EGFR protein levels after SOF treatment were identified by immunoblot analysis. One representative immunoblot is displayed. EGFR expression is presented in the bar graphs as a fold change relative to the DMSO control (mean + s.d. from three biological replicates). ( c ) PHHs were incubated with rising concentrations of SOF for four days. EGFR and B-MYB protein levels were evaluated by immunoblot analysis. One representative immunoblot of two independent experiment is displayed. ( d ) HepG2 and ( e ) HEK-293 cells were starved for 24 h prior to SOF treatment. Protein levels of the phosphorylated form of EGFR (pEGFR) were assessed at the depicted time points by immunoblot analysis. One representative immunoblot is presented. pEGFR expression is shown as a fold change in relation to time point 0 (mean + s.d. from three biological replicates). ( f ) EGFR, pEGFR, B-MYB, and Cyclin D1 protein levels after treatment with SOF and SOF in combination with the EGFR inhibitor, erlotinib (ERL), were identified by immunoblot analysis. One representative immunoblot of two independent experiments is shown. Statistical significance was determined through an unpaired t -test ( a , b ), paired t -test (d, time point 0 vs. 240 min), and two-way ANOVA ( d , e ). ns: not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005.
    Figure Legend Snippet: Sofosbuvir increases the expression and activation of EGFR. ( a , b ) EGFR protein levels after SOF treatment were identified by immunoblot analysis. One representative immunoblot is displayed. EGFR expression is presented in the bar graphs as a fold change relative to the DMSO control (mean + s.d. from three biological replicates). ( c ) PHHs were incubated with rising concentrations of SOF for four days. EGFR and B-MYB protein levels were evaluated by immunoblot analysis. One representative immunoblot of two independent experiment is displayed. ( d ) HepG2 and ( e ) HEK-293 cells were starved for 24 h prior to SOF treatment. Protein levels of the phosphorylated form of EGFR (pEGFR) were assessed at the depicted time points by immunoblot analysis. One representative immunoblot is presented. pEGFR expression is shown as a fold change in relation to time point 0 (mean + s.d. from three biological replicates). ( f ) EGFR, pEGFR, B-MYB, and Cyclin D1 protein levels after treatment with SOF and SOF in combination with the EGFR inhibitor, erlotinib (ERL), were identified by immunoblot analysis. One representative immunoblot of two independent experiments is shown. Statistical significance was determined through an unpaired t -test ( a , b ), paired t -test (d, time point 0 vs. 240 min), and two-way ANOVA ( d , e ). ns: not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005.

    Techniques Used: Expressing, Activation Assay, Incubation

    Regulation of EGFR phosphorylation and nuclear translocation during SOF treatment. ( a ) HepG2 cells were starved for 24 h and subsequently treated with SOF alone or in combination with pEGFR inhibitor (ERL) or p-p38 inhibitor (DOR). The protein level of target proteins was assessed at depicted time points by immunoblot analysis. One representative immunoblot is presented. Bar graphs display the relative quantification of pEGFR and p-p38 shown as fold change in relation to time point 0 (mean + s.d. from three independent experiments). Statistical significance was determined through two-way ANOVA (DMSO vs. treatment). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005. ( b ) The expression of target proteins in the cytoplasm and nucleus after SOF treatment at different time points was detected by immunoblot an lysis. One representative immunoblot of three (EGFR, pEGFR) and two (p-p38, p38) independent experiments is shown.
    Figure Legend Snippet: Regulation of EGFR phosphorylation and nuclear translocation during SOF treatment. ( a ) HepG2 cells were starved for 24 h and subsequently treated with SOF alone or in combination with pEGFR inhibitor (ERL) or p-p38 inhibitor (DOR). The protein level of target proteins was assessed at depicted time points by immunoblot analysis. One representative immunoblot is presented. Bar graphs display the relative quantification of pEGFR and p-p38 shown as fold change in relation to time point 0 (mean + s.d. from three independent experiments). Statistical significance was determined through two-way ANOVA (DMSO vs. treatment). * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005. ( b ) The expression of target proteins in the cytoplasm and nucleus after SOF treatment at different time points was detected by immunoblot an lysis. One representative immunoblot of three (EGFR, pEGFR) and two (p-p38, p38) independent experiments is shown.

    Techniques Used: Translocation Assay, Expressing, Lysis

    Effect of different nucleotide analogues on alteration in the cell phenotype and activation of EGFR. ( a ) Cytotoxicity of zidovudine (AZT) and tenofovir (TDF) in HepG2 cells was detected by Rotitest ® Vital. Bar graph displays absorbance as a fold change in relation to DMSO. ( b ) Cell cycle analysis of HepG2 cells treated with AZT and TDF. ( c ) Apoptosis induction was evaluated with Annexin V and live/dead cell staining by flow cytometry. ( d ) Proliferation rates were determined by trypan blue exclusion and displayed in relation to DMSO. ( e ) EGFR expression after four days of continuous treatment with AZT and TDF. One representative immunoblot is shown. Bar graph presents relative quantification of EGFR as a fold change compared to DMSO. ( f ) Activation of EGFR after AZT and TDF therapy was evaluated by immunoblot analysis at different time points. One representative immunoblot is depicted. pEGFR expression is shown as a fold change in relation to time point 0. All graphs present mean + s.d. from three independent experiments. Statistical significance was determined through one-way ANOVA ( a , c , d ) and two-way ANOVA ( b , f ). ns: not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005.
    Figure Legend Snippet: Effect of different nucleotide analogues on alteration in the cell phenotype and activation of EGFR. ( a ) Cytotoxicity of zidovudine (AZT) and tenofovir (TDF) in HepG2 cells was detected by Rotitest ® Vital. Bar graph displays absorbance as a fold change in relation to DMSO. ( b ) Cell cycle analysis of HepG2 cells treated with AZT and TDF. ( c ) Apoptosis induction was evaluated with Annexin V and live/dead cell staining by flow cytometry. ( d ) Proliferation rates were determined by trypan blue exclusion and displayed in relation to DMSO. ( e ) EGFR expression after four days of continuous treatment with AZT and TDF. One representative immunoblot is shown. Bar graph presents relative quantification of EGFR as a fold change compared to DMSO. ( f ) Activation of EGFR after AZT and TDF therapy was evaluated by immunoblot analysis at different time points. One representative immunoblot is depicted. pEGFR expression is shown as a fold change in relation to time point 0. All graphs present mean + s.d. from three independent experiments. Statistical significance was determined through one-way ANOVA ( a , c , d ) and two-way ANOVA ( b , f ). ns: not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.005.

    Techniques Used: Activation Assay, Cell Cycle Assay, Staining, Flow Cytometry, Expressing

    6) Product Images from "Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype"

    Article Title: Interaction of glycosphingolipids GD3 and GD2 with growth factor receptors maintains breast cancer stem cell phenotype

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17665

    Colocalization of GD2 and GD3 with GFRs in GD3S-overexpressing breast cancer cell lines ( A – C ) GD3S-overexpressing MDA-MB231, MDA-MB468, and MCF7 cells were fixed, permeabilized, and then stained for immunofluorescence (left images) by anti-GD3 (red)/ anti-EGFR (green) or anti-GD2 (red)/ anti-c-Met (green). Nuclei were stained with DAPI (blue). Colocalization signals are shown as yellow in merged images. GD3/EGFR and GD2/c-Met associations were further investigated by in situ PLA (right images). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 20 or 40 μm.
    Figure Legend Snippet: Colocalization of GD2 and GD3 with GFRs in GD3S-overexpressing breast cancer cell lines ( A – C ) GD3S-overexpressing MDA-MB231, MDA-MB468, and MCF7 cells were fixed, permeabilized, and then stained for immunofluorescence (left images) by anti-GD3 (red)/ anti-EGFR (green) or anti-GD2 (red)/ anti-c-Met (green). Nuclei were stained with DAPI (blue). Colocalization signals are shown as yellow in merged images. GD3/EGFR and GD2/c-Met associations were further investigated by in situ PLA (right images). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 20 or 40 μm.

    Techniques Used: Multiple Displacement Amplification, Staining, Immunofluorescence, In Situ, Proximity Ligation Assay

    Association of GD2 and GD3 with GFRs in breast CSCs Association between GD2/GD3 and GFRs was evaluated using Twist-induced EMT of HMLE-Twist-ER cells (breast CSCs). ( A ) Cell lysates of breast CSCs were immunoprecipitated (IP) with mouse anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG, followed by Western blotting (WB). WB was probed with anti-EGFR (left panel), anti-c-Met (middle panel), or anti-integrin β1 (right panel) mAb to detect components of Ab-absorbed complexes. Integrin β1/GD2 association was used as positive control. ( B ) Semi-confluent monolayers of breast CSCs were fixed and permeabilized. Cells were immune-stained with anti-GD3 (red) and anti-EGFR (green) (upper panels) or with anti-GD2 (red) and anti-c-Met (green) (lower panels), for immunofluorescence double labeling in combination with DAPI staining (blue) for cell nuclei. Scale bar = 10 μm. ( C ) GD3/EGFR and GD2/c-Met associations were evaluated by immunogold-TEM. Gangliosides (GD2, GD3) were probed with 12 nm colloidal gold particles; GFRs (EGFR, c-Met) were probed with 6 nm colloidal gold particles. Scale bar = 100 or 25 nm. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 100 or 25 nm. ( D ) GD3/EGFR and GD2/c-Met associations in breast CSCs were investigated by in situ proximity ligation assay (PLA). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Cell nuclei were stained with DAPI (blue). Scale bar = 20 μm.
    Figure Legend Snippet: Association of GD2 and GD3 with GFRs in breast CSCs Association between GD2/GD3 and GFRs was evaluated using Twist-induced EMT of HMLE-Twist-ER cells (breast CSCs). ( A ) Cell lysates of breast CSCs were immunoprecipitated (IP) with mouse anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG, followed by Western blotting (WB). WB was probed with anti-EGFR (left panel), anti-c-Met (middle panel), or anti-integrin β1 (right panel) mAb to detect components of Ab-absorbed complexes. Integrin β1/GD2 association was used as positive control. ( B ) Semi-confluent monolayers of breast CSCs were fixed and permeabilized. Cells were immune-stained with anti-GD3 (red) and anti-EGFR (green) (upper panels) or with anti-GD2 (red) and anti-c-Met (green) (lower panels), for immunofluorescence double labeling in combination with DAPI staining (blue) for cell nuclei. Scale bar = 10 μm. ( C ) GD3/EGFR and GD2/c-Met associations were evaluated by immunogold-TEM. Gangliosides (GD2, GD3) were probed with 12 nm colloidal gold particles; GFRs (EGFR, c-Met) were probed with 6 nm colloidal gold particles. Scale bar = 100 or 25 nm. Regions indicated by white boxes are shown at higher magnification in lower panels. Scale bar = 100 or 25 nm. ( D ) GD3/EGFR and GD2/c-Met associations in breast CSCs were investigated by in situ proximity ligation assay (PLA). Each PLA signal is visualized as a red fluorescent spot, and represents one detected association event. Cell nuclei were stained with DAPI (blue). Scale bar = 20 μm.

    Techniques Used: Immunoprecipitation, Western Blot, Positive Control, Staining, Immunofluorescence, Labeling, Transmission Electron Microscopy, In Situ, Proximity Ligation Assay

    7) Product Images from "Tanshinone IIA Reverses Gefitinib-Resistance In Human Non-Small-Cell Lung Cancer Via Regulation Of VEGFR/Akt Pathway"

    Article Title: Tanshinone IIA Reverses Gefitinib-Resistance In Human Non-Small-Cell Lung Cancer Via Regulation Of VEGFR/Akt Pathway

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S221228

    Tan IIA enhances the sensitivity of HCC827/gefitinib cells to gefitinib by regulation of VEGFR/Akt pathway. HCC827/gefitinib cells were treated with 40 nM OXA or/and 2 μM Tan IIA, or OXA + Tan IIA + VEGF for 72 hrs. ( A ) Expression levels of p-EGFR, p-VEGFR2 and p-Akt in HCC827/gefitinib cells were detected with Western blotting. β-actin was used as an internal control. ( B–D ) The relative expressions of p-EGFR, p-VEGFR2 and p-Akt in HCC827/gefitinib cells were quantified via normalization to β-actin. *P
    Figure Legend Snippet: Tan IIA enhances the sensitivity of HCC827/gefitinib cells to gefitinib by regulation of VEGFR/Akt pathway. HCC827/gefitinib cells were treated with 40 nM OXA or/and 2 μM Tan IIA, or OXA + Tan IIA + VEGF for 72 hrs. ( A ) Expression levels of p-EGFR, p-VEGFR2 and p-Akt in HCC827/gefitinib cells were detected with Western blotting. β-actin was used as an internal control. ( B–D ) The relative expressions of p-EGFR, p-VEGFR2 and p-Akt in HCC827/gefitinib cells were quantified via normalization to β-actin. *P

    Techniques Used: Expressing, Western Blot

    Tan IIA enhances the sensitivity of HCC827/gefitinib cells to gefitinib in vivo. HCC827/gefitinib cells were injected subcutaneously into nude mice to form subcutaneous tumors. ( A ) Tumor volumes of mice were measured weekly. ( B ) HCC827/gefitinib xenograft tumors were excised from xenografts and pictured on day 21. ( C ) The weight in each group of mice was calculated on day 21. ( D ) Expression levels of p-EGFR, p-VEGFR2 and p-Akt in tumor tissues were measured with Western blotting. ( E, F, G ) The relative expressions of p-EGFR, p-VEGFR2 and p-Akt were quantified via normalization to β-actin. *P
    Figure Legend Snippet: Tan IIA enhances the sensitivity of HCC827/gefitinib cells to gefitinib in vivo. HCC827/gefitinib cells were injected subcutaneously into nude mice to form subcutaneous tumors. ( A ) Tumor volumes of mice were measured weekly. ( B ) HCC827/gefitinib xenograft tumors were excised from xenografts and pictured on day 21. ( C ) The weight in each group of mice was calculated on day 21. ( D ) Expression levels of p-EGFR, p-VEGFR2 and p-Akt in tumor tissues were measured with Western blotting. ( E, F, G ) The relative expressions of p-EGFR, p-VEGFR2 and p-Akt were quantified via normalization to β-actin. *P

    Techniques Used: In Vivo, Injection, Mouse Assay, Expressing, Western Blot

    8) Product Images from "Selective uptake of epidermal growth factor-conjugated gold nanoparticle (EGF-GNP) facilitates non-thermal plasma (NTP)-mediated cell death"

    Article Title: Selective uptake of epidermal growth factor-conjugated gold nanoparticle (EGF-GNP) facilitates non-thermal plasma (NTP)-mediated cell death

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11292-z

    A549, human lung carcinoma cell, expresses a high level of EGFR. ( A ) A549, DU145, HeLa, HT29, SH-SY5Y, and SK-OV (3) cells were cultured and whole-cell extracts were analyzed by Western blotting for EGFR assessment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) A549 cells were cultured in 35 mm 2 dishes until 80~90% of confluency. NTP was generated at different voltages and cells were exposed for different time periods as indicated in the figure. Each value represents the mean ± S.D. of triplicate samples. ( C ) Trypan blue staining was performed to assess dead cell population after plasma treatment. Exposure time and condition are indicated in the figure.
    Figure Legend Snippet: A549, human lung carcinoma cell, expresses a high level of EGFR. ( A ) A549, DU145, HeLa, HT29, SH-SY5Y, and SK-OV (3) cells were cultured and whole-cell extracts were analyzed by Western blotting for EGFR assessment. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) A549 cells were cultured in 35 mm 2 dishes until 80~90% of confluency. NTP was generated at different voltages and cells were exposed for different time periods as indicated in the figure. Each value represents the mean ± S.D. of triplicate samples. ( C ) Trypan blue staining was performed to assess dead cell population after plasma treatment. Exposure time and condition are indicated in the figure.

    Techniques Used: Cell Culture, Western Blot, Generated, Staining

    9) Product Images from "Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids"

    Article Title: Lactobacillus rhamnosus GG prevents epithelial barrier dysfunction induced by interferon-gamma and fecal supernatants from irritable bowel syndrome patients in human intestinal enteroids and colonoids

    Journal: Gut Microbes

    doi: 10.1080/19490976.2018.1479625

    Protective actions of LGG on junction proteins were mediated by pathways independent of MAPK/ERK signaling cascade in human colonoids . Human colonoids were treated with LGG-CM or EGF (100 ng/ml) for 90 min in the presence or absence of 1-h pretreatment with an EGFR inhibitor, AG1478 (200nM). In separate experiments, human colonoids were treated with IFN-gamma (200 ng/ml) in the absence or presence of LGG. The protective action of LGG was examined in the presence of AG 1478 (200nM). Cellular lysates were prepared and immunoblotted for P-EGFR, total EGFR, P-ERK, total ERK, occludin, ZO-1 and GAPDH. Bands of GAPDH were used as control for an equal protein loading. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE; n = 5 in each group (control group vs EGF group *p
    Figure Legend Snippet: Protective actions of LGG on junction proteins were mediated by pathways independent of MAPK/ERK signaling cascade in human colonoids . Human colonoids were treated with LGG-CM or EGF (100 ng/ml) for 90 min in the presence or absence of 1-h pretreatment with an EGFR inhibitor, AG1478 (200nM). In separate experiments, human colonoids were treated with IFN-gamma (200 ng/ml) in the absence or presence of LGG. The protective action of LGG was examined in the presence of AG 1478 (200nM). Cellular lysates were prepared and immunoblotted for P-EGFR, total EGFR, P-ERK, total ERK, occludin, ZO-1 and GAPDH. Bands of GAPDH were used as control for an equal protein loading. The optical density is expressed in arbitrary units normalized against a control sample. Data in histograms represent means ± SE; n = 5 in each group (control group vs EGF group *p

    Techniques Used:

    10) Product Images from "Expression of LRIG1, a Negative Regulator of EGFR, Is Dynamically Altered during Different Stages of Gastric Carcinogenesis"

    Article Title: Expression of LRIG1, a Negative Regulator of EGFR, Is Dynamically Altered during Different Stages of Gastric Carcinogenesis

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.08.006

    Immunohistochemistry (IHC) analysis of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 and epidermal growth factor receptor (EGFR) expression in human gastric preneoplastic and cancer tissues. LRIG1 and EGFR were detected in human gastric preneoplastic and cancer tissues using IHC with indicated antibodies. A and B: In a spasmolytic polypeptide–expressing metaplastic (SPEM) region ( A ), LRIG1 was expressed strongly, whereas EGFR expression was low along SPEM glands ( arrowheads ). C–E: Decreased LRIG1 expression ( C and D ; yellow arrowheads ) and low EGFR gland expression ( C and D ; black arrowheads ) are observed in the intestinal metaplastic (IM) region, and LRIG1 is absent in cancer tissues ( E ). D and F: Strong EGFR expression in the IM region ( D , yellow arrowheads ) and in cancer lesions ( F ) is shown. Samples from five patients were used in these experiments G: LRIG1- or EGFR-positive cells were counted from 10 microscopic fields. Statistical analyses were performed using JMP software version 4. Data are expressed as means ± SEM. ∗ P
    Figure Legend Snippet: Immunohistochemistry (IHC) analysis of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 and epidermal growth factor receptor (EGFR) expression in human gastric preneoplastic and cancer tissues. LRIG1 and EGFR were detected in human gastric preneoplastic and cancer tissues using IHC with indicated antibodies. A and B: In a spasmolytic polypeptide–expressing metaplastic (SPEM) region ( A ), LRIG1 was expressed strongly, whereas EGFR expression was low along SPEM glands ( arrowheads ). C–E: Decreased LRIG1 expression ( C and D ; yellow arrowheads ) and low EGFR gland expression ( C and D ; black arrowheads ) are observed in the intestinal metaplastic (IM) region, and LRIG1 is absent in cancer tissues ( E ). D and F: Strong EGFR expression in the IM region ( D , yellow arrowheads ) and in cancer lesions ( F ) is shown. Samples from five patients were used in these experiments G: LRIG1- or EGFR-positive cells were counted from 10 microscopic fields. Statistical analyses were performed using JMP software version 4. Data are expressed as means ± SEM. ∗ P

    Techniques Used: Immunohistochemistry, Expressing, Software

    Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on tumorigenesis in the SNU638 gastric cancer cell line. A: LRIG1 was depleted using pGIPZ-based shRNA (sh46 and sh71). Levels of LRIG1, epidermal growth factor receptor (EGFR), phospho-EGFR, and epithelial to mesenchymal markers [E-cadherin, occludin, N-cadherin, vimentin, zinc finger E box–binding homeobox (ZEB)-1/T-cell factor (TCF)-3, and anti–zinc finger protein SNAI1 (SNAIL)] were detected by Western blot analysis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. B: Cell proliferation was assessed with cell counting over 5 days. C: Cell cycle was examined with flow cytometry. D: Representative image of invasion and migration analyzed using Boyden chambers. E: Quantitative-analytic data from four independent experiments. Data are expressed as means ± SEM. n = 3 ( C ); n = 4 ( A , B , and D ). ∗ P
    Figure Legend Snippet: Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on tumorigenesis in the SNU638 gastric cancer cell line. A: LRIG1 was depleted using pGIPZ-based shRNA (sh46 and sh71). Levels of LRIG1, epidermal growth factor receptor (EGFR), phospho-EGFR, and epithelial to mesenchymal markers [E-cadherin, occludin, N-cadherin, vimentin, zinc finger E box–binding homeobox (ZEB)-1/T-cell factor (TCF)-3, and anti–zinc finger protein SNAI1 (SNAIL)] were detected by Western blot analysis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. B: Cell proliferation was assessed with cell counting over 5 days. C: Cell cycle was examined with flow cytometry. D: Representative image of invasion and migration analyzed using Boyden chambers. E: Quantitative-analytic data from four independent experiments. Data are expressed as means ± SEM. n = 3 ( C ); n = 4 ( A , B , and D ). ∗ P

    Techniques Used: shRNA, Binding Assay, Western Blot, Cell Counting, Flow Cytometry, Cytometry, Migration

    Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on SNU638 xenografts and Kaplan-Meier survival curves of relapse-free survival (RFS) of gastric cancer patients with low or high LRIG1 mRNA expression. SNU638 cells, with or without vector transfection, were injected s.c. into the dorsal flank of 7-week-old male athymic nude mice to establish primary tumors. A: Tumor volumes were measured using calipers. B: Extent of tumor growth was evaluated in vivo . Dotted circles represent tumor mass. C: Histopathologic analysis of gastric tumor masses from mouse xenograft model. Hematoxylin and eosin (H E) staining was performed to investigate epidermal growth factor receptor (EGFR) and phospho (p)-EGFR (Tyr1068) expression in tumor tissues from xenograft mice. Boxed areas are shown at higher magnification to the right. EGFR- and pEGFR-positive cells were counted from five microscopic fields. Statistical analyses were performed using JMP software version 4. D: Kaplan-Meier curves show RFS in gastric cancer patients, monitored for 150 months, based on high or low tumor LRIG1 mRNA expression levels. Red, patients with expression levels above the median; black, patients with expression levels below the median. Patients with low expression levels had a lower probability of RFS over time. Data are expressed as means ± SEM. n = 4 mice per treatment. ∗ P
    Figure Legend Snippet: Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on SNU638 xenografts and Kaplan-Meier survival curves of relapse-free survival (RFS) of gastric cancer patients with low or high LRIG1 mRNA expression. SNU638 cells, with or without vector transfection, were injected s.c. into the dorsal flank of 7-week-old male athymic nude mice to establish primary tumors. A: Tumor volumes were measured using calipers. B: Extent of tumor growth was evaluated in vivo . Dotted circles represent tumor mass. C: Histopathologic analysis of gastric tumor masses from mouse xenograft model. Hematoxylin and eosin (H E) staining was performed to investigate epidermal growth factor receptor (EGFR) and phospho (p)-EGFR (Tyr1068) expression in tumor tissues from xenograft mice. Boxed areas are shown at higher magnification to the right. EGFR- and pEGFR-positive cells were counted from five microscopic fields. Statistical analyses were performed using JMP software version 4. D: Kaplan-Meier curves show RFS in gastric cancer patients, monitored for 150 months, based on high or low tumor LRIG1 mRNA expression levels. Red, patients with expression levels above the median; black, patients with expression levels below the median. Patients with low expression levels had a lower probability of RFS over time. Data are expressed as means ± SEM. n = 4 mice per treatment. ∗ P

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Injection, Mouse Assay, In Vivo, Staining, Software

    11) Product Images from "A novel co-drug of aspirin and ursolic acid interrupts adhesion, invasion and migration of cancer cells to vascular endothelium via regulating EMT and EGFR-mediated signaling pathways: multiple targets for cancer metastasis prevention and treatment"

    Article Title: A novel co-drug of aspirin and ursolic acid interrupts adhesion, invasion and migration of cancer cells to vascular endothelium via regulating EMT and EGFR-mediated signaling pathways: multiple targets for cancer metastasis prevention and treatment

    Journal: Oncotarget

    doi: 10.18632/oncotarget.12232

    Influence of Asp-UA on the cell invasion molecules, EMT and EGFR related cell signaling pathways ( A ) Western blot analysis of UA/Asp/Asp-UA on the expression of MMP-2, MMP-9 and COX-2 in MCF-7 cells and ( B ) MDA-MB-231 cells. ( C ) Expression of E-cadherin on MCF-7 cells was determined by flow cytometry, isotype control (gray area), control (black curve), blue curve (15 μM UA), yellow curve (1 mM Asp) and red line represents Asp-UA treated group at concentrations of 20 and 40 μM, respectively. ( D ) Western blotting analysis of UA/Asp/Asp-UA on the regulation of E-cadherin, β-catenin, EGFR, ERK and PTEN in MCF-7 ( E ) and MDA-MB-231 cells ( F ). The inhibitory effects of UA, Asp and Asp-UA on the expression of E-cadherin by FACS. ( G ) Double immunofluorescence staining with DAPI (blue) and anti-EGFR antibody (red) was carried out in MCF-7 cells treated with Asp-UA for 24 h. The data were obtained from 3 separate experiments and bars represent the mean ± SD. (* P
    Figure Legend Snippet: Influence of Asp-UA on the cell invasion molecules, EMT and EGFR related cell signaling pathways ( A ) Western blot analysis of UA/Asp/Asp-UA on the expression of MMP-2, MMP-9 and COX-2 in MCF-7 cells and ( B ) MDA-MB-231 cells. ( C ) Expression of E-cadherin on MCF-7 cells was determined by flow cytometry, isotype control (gray area), control (black curve), blue curve (15 μM UA), yellow curve (1 mM Asp) and red line represents Asp-UA treated group at concentrations of 20 and 40 μM, respectively. ( D ) Western blotting analysis of UA/Asp/Asp-UA on the regulation of E-cadherin, β-catenin, EGFR, ERK and PTEN in MCF-7 ( E ) and MDA-MB-231 cells ( F ). The inhibitory effects of UA, Asp and Asp-UA on the expression of E-cadherin by FACS. ( G ) Double immunofluorescence staining with DAPI (blue) and anti-EGFR antibody (red) was carried out in MCF-7 cells treated with Asp-UA for 24 h. The data were obtained from 3 separate experiments and bars represent the mean ± SD. (* P

    Techniques Used: Western Blot, Expressing, Multiple Displacement Amplification, Flow Cytometry, Cytometry, FACS, Double Immunofluorescence Staining

    12) Product Images from "Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness"

    Article Title: Multidrug transporter MRP4/ABCC4 as a key determinant of pancreatic cancer aggressiveness

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-71181-w

    MRP4 silencing reduces tumor growth and tumorigenicity in PANC1 xenografts. Swiss nude mice were subcutaneously injected either with PANC1-MRP4sh2, PANC1-MRP4sh1 clones or PANC1-scramble as a control. Each group contained 8–11 animals and the data shown represents measurements of one of two experiments. ( a ; left) Tumors were measured with a caliper three times a week for 32 days post-inoculation and tumor volume was calculated as described in “ Materials and Methods ”. ( a ; middle) Representative pictures of the dissected xenografts of each group at the end of the experiment. ( a ; right) Tumor weight was measured with a scale after dissection. ( b ) PANC1-scramble, PANC1-MRP4sh2 and PANC1-MRP4sh1 xenografts were processed for histological (H E staining), MRP4, Ki67, and EGFR immunostaining evaluation. Nuclei were counterstained with hematoxylin. Representative images are shown. Cell number per field, proliferative index, MRP4 and EGFR scores were determined by H E, Ki67, MRP4, and EGFR staining respectively, and are shown as bar plots (right). Data is shown as mean ± SEM. Statistics: Linear regression and one-way ANOVA followed by Tukey’s t -test. *** p
    Figure Legend Snippet: MRP4 silencing reduces tumor growth and tumorigenicity in PANC1 xenografts. Swiss nude mice were subcutaneously injected either with PANC1-MRP4sh2, PANC1-MRP4sh1 clones or PANC1-scramble as a control. Each group contained 8–11 animals and the data shown represents measurements of one of two experiments. ( a ; left) Tumors were measured with a caliper three times a week for 32 days post-inoculation and tumor volume was calculated as described in “ Materials and Methods ”. ( a ; middle) Representative pictures of the dissected xenografts of each group at the end of the experiment. ( a ; right) Tumor weight was measured with a scale after dissection. ( b ) PANC1-scramble, PANC1-MRP4sh2 and PANC1-MRP4sh1 xenografts were processed for histological (H E staining), MRP4, Ki67, and EGFR immunostaining evaluation. Nuclei were counterstained with hematoxylin. Representative images are shown. Cell number per field, proliferative index, MRP4 and EGFR scores were determined by H E, Ki67, MRP4, and EGFR staining respectively, and are shown as bar plots (right). Data is shown as mean ± SEM. Statistics: Linear regression and one-way ANOVA followed by Tukey’s t -test. *** p

    Techniques Used: Mouse Assay, Injection, Clone Assay, Dissection, Staining, Immunostaining

    MRP4 overexpression increases tumor growth in BxPC-3 xenografts. NSG mice were subcutaneously injected with BxPC-3-MRP4 or BxPC-3-mock as a control. Each group contained 5 animals and the data shows measurements of one of two experiments. ( a ; left) Tumors were measured with a caliper three times a week for 32 days post-inoculation and tumor volume was calculated as described in “ Materials and methods ”. ( a ; middle) Representative pictures of the dissected xenografts of each group at the end of the experiment. ( a ; right) Tumor weight was measured with a scale after tumor dissection. ( b ) BxPC-3-mock and BxPC-3-MRP4 xenografts were processed for histological (H E staining), MRP4, Ki67, and EGFR immunostaining evaluation. Nuclei were counterstained with hematoxylin. Representative images are shown. Cell number per field, proliferative index, MRP4 and EGFR scores were determined by H E, Ki67, MRP4, and EGFR staining respectively, and are shown as bar plots (right). Data is shown as mean ± SEM. Statistics: Linear regression and Student´s t -test. *** p
    Figure Legend Snippet: MRP4 overexpression increases tumor growth in BxPC-3 xenografts. NSG mice were subcutaneously injected with BxPC-3-MRP4 or BxPC-3-mock as a control. Each group contained 5 animals and the data shows measurements of one of two experiments. ( a ; left) Tumors were measured with a caliper three times a week for 32 days post-inoculation and tumor volume was calculated as described in “ Materials and methods ”. ( a ; middle) Representative pictures of the dissected xenografts of each group at the end of the experiment. ( a ; right) Tumor weight was measured with a scale after tumor dissection. ( b ) BxPC-3-mock and BxPC-3-MRP4 xenografts were processed for histological (H E staining), MRP4, Ki67, and EGFR immunostaining evaluation. Nuclei were counterstained with hematoxylin. Representative images are shown. Cell number per field, proliferative index, MRP4 and EGFR scores were determined by H E, Ki67, MRP4, and EGFR staining respectively, and are shown as bar plots (right). Data is shown as mean ± SEM. Statistics: Linear regression and Student´s t -test. *** p

    Techniques Used: Over Expression, Mouse Assay, Injection, Dissection, Staining, Immunostaining

    Bioinformatic analysis of MRP4 differential expression in PDAC samples, normal/adjacent pancreatic tissue, PDAC cells lines and circulating tumor cells from PDAC patients. ( a ) Differential expression of MRP4 according to the Gene Expression Profiling Interactive Analysis (GEPIA) database using TCGA PAAD tumor data and matched data of normal tissue from TCGA. ( b ) Comparative analysis of MRP4 mRNA expression from three different gene expression datasets comprising pancreatic normal tissue and their paired PDAC samples (GSE15471 and GSE62452), as well as PDAC cell lines (GSE71729). ( c ) MRP4, vimentin, E-cadherin, and EGFR transcript levels in a panel of 9 pancreatic cancer cell lines systematically arranged from the less aggressive (Lo-G) to the more aggressive (Hi-G) phenotypes (GSE64557). ( d ) Inverse correlation between MRP4 and GATA6 expression levels in the 9 pancreatic cancer cell lines from the beforementioned dataset. ( e ) Heatmap showing differentially expressed genes in MRP4 LE and HE PDAC samples performed with MultiExperiment Viewer (MeV). Differentially expressed genes were identified based on a log2(fold change) ≥ 1 and an FDR ≤ 0.05. Hierarchical clustering was based on Pearson correlation coefficients. ( f ) Functional enrichment analysis of differentially expressed genes showing pathways associated with MRP4 expression (p-adjusted
    Figure Legend Snippet: Bioinformatic analysis of MRP4 differential expression in PDAC samples, normal/adjacent pancreatic tissue, PDAC cells lines and circulating tumor cells from PDAC patients. ( a ) Differential expression of MRP4 according to the Gene Expression Profiling Interactive Analysis (GEPIA) database using TCGA PAAD tumor data and matched data of normal tissue from TCGA. ( b ) Comparative analysis of MRP4 mRNA expression from three different gene expression datasets comprising pancreatic normal tissue and their paired PDAC samples (GSE15471 and GSE62452), as well as PDAC cell lines (GSE71729). ( c ) MRP4, vimentin, E-cadherin, and EGFR transcript levels in a panel of 9 pancreatic cancer cell lines systematically arranged from the less aggressive (Lo-G) to the more aggressive (Hi-G) phenotypes (GSE64557). ( d ) Inverse correlation between MRP4 and GATA6 expression levels in the 9 pancreatic cancer cell lines from the beforementioned dataset. ( e ) Heatmap showing differentially expressed genes in MRP4 LE and HE PDAC samples performed with MultiExperiment Viewer (MeV). Differentially expressed genes were identified based on a log2(fold change) ≥ 1 and an FDR ≤ 0.05. Hierarchical clustering was based on Pearson correlation coefficients. ( f ) Functional enrichment analysis of differentially expressed genes showing pathways associated with MRP4 expression (p-adjusted

    Techniques Used: Expressing, Functional Assay

    Effect of MRP4 overexpression upon BxPC-3 experimental metastasis. ( a ) Vimentin and E-cadherin (CDH1) transcript levels in BxPC-3-mock and BxPC-3-MRP4 cells, determined by qPCR as described in “ Materials and methods ”. Data is shown as mean ± SD of three measurements and the experiment was performed three times. ( b ) H E staining, GFP, Ki67, and EGFR immunostaining of hepatic, renal, and pulmonary metastatic foci from BxPC-3-MRP4 inoculated mice. Nuclei were counterstained with hematoxylin. Representative images are shown. ( c ) Representative liver macro-metastases at the end of the experiment. ( d ) Representative comparison of GFP expression in liver homogenates, determined by FAC. ( e ) Number of hepatic metastatic foci per animal in each experimental group. Data is shown as mean ± SEM. Statistics: Student’s t -test. *** p
    Figure Legend Snippet: Effect of MRP4 overexpression upon BxPC-3 experimental metastasis. ( a ) Vimentin and E-cadherin (CDH1) transcript levels in BxPC-3-mock and BxPC-3-MRP4 cells, determined by qPCR as described in “ Materials and methods ”. Data is shown as mean ± SD of three measurements and the experiment was performed three times. ( b ) H E staining, GFP, Ki67, and EGFR immunostaining of hepatic, renal, and pulmonary metastatic foci from BxPC-3-MRP4 inoculated mice. Nuclei were counterstained with hematoxylin. Representative images are shown. ( c ) Representative liver macro-metastases at the end of the experiment. ( d ) Representative comparison of GFP expression in liver homogenates, determined by FAC. ( e ) Number of hepatic metastatic foci per animal in each experimental group. Data is shown as mean ± SEM. Statistics: Student’s t -test. *** p

    Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Staining, Immunostaining, Mouse Assay, Expressing

    13) Product Images from "The Effects of Artesunate on the Expression of EGFR and ABCG2 in A549 Human Lung Cancer Cells and a Xenograft Model"

    Article Title: The Effects of Artesunate on the Expression of EGFR and ABCG2 in A549 Human Lung Cancer Cells and a Xenograft Model

    Journal: Molecules

    doi: 10.3390/molecules161210556

    The expression of ABCG2, Akt, p-Akt and p-EGFR proteins was examined in untreated and ART-treated (60 mg·kg −1 and 120 mg·kg −1 ) xenografts. Photographs of immunohistochemical staining for ABCG2, Akt, p-Akt and p-EGFR (×40) are shown. A distinct yellow stain can be observed in the membrane and cytoplasm. The images presented are representative of three independent experiments. The data are the means ± SE from three independent experiments performed in triplicate. * p
    Figure Legend Snippet: The expression of ABCG2, Akt, p-Akt and p-EGFR proteins was examined in untreated and ART-treated (60 mg·kg −1 and 120 mg·kg −1 ) xenografts. Photographs of immunohistochemical staining for ABCG2, Akt, p-Akt and p-EGFR (×40) are shown. A distinct yellow stain can be observed in the membrane and cytoplasm. The images presented are representative of three independent experiments. The data are the means ± SE from three independent experiments performed in triplicate. * p

    Techniques Used: Expressing, Immunohistochemistry, Staining

    ART down-regulates the EGFR and ABCG2 mRNA and protein levels in A549 cells. ( A ) Cells were treated with varying concentrations of ART for 48 h and then harvested. The EGFR and ABCG2 mRNA levels were detected using real-time RT-PCR; ( B ) The expression of EGFR, p-EGFR Akt, p-Akt and ABCG2 was measured by western blot analysis. A549 cells were treated with varying concentrations of ART for 48 h, then β-actin expression was used as an internal control to determine the expression levels of proteins. Significant down-regulation of all proteins was observed. The blots are representative of three independent experiments. The data represent the means ± SE from three independent experiments performed in triplicate. * p
    Figure Legend Snippet: ART down-regulates the EGFR and ABCG2 mRNA and protein levels in A549 cells. ( A ) Cells were treated with varying concentrations of ART for 48 h and then harvested. The EGFR and ABCG2 mRNA levels were detected using real-time RT-PCR; ( B ) The expression of EGFR, p-EGFR Akt, p-Akt and ABCG2 was measured by western blot analysis. A549 cells were treated with varying concentrations of ART for 48 h, then β-actin expression was used as an internal control to determine the expression levels of proteins. Significant down-regulation of all proteins was observed. The blots are representative of three independent experiments. The data represent the means ± SE from three independent experiments performed in triplicate. * p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

    14) Product Images from "The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion"

    Article Title: The long noncoding RNA lnc-EGFR stimulates T-regulatory cells differentiation thus promoting hepatocellular carcinoma immune evasion

    Journal: Nature Communications

    doi: 10.1038/ncomms15129

    Cytoplasmic lnc-EGFR binds specifically to EGFR. ( a ) Lnc-EGFR RNA pull-down assay was performed. The associated proteins were processed and subjected to Mass Spec. followed by analysis via the Proteome Discoverer program (a1) and the NCBI protein database with the Mascot search engine (a2). ( b ) RNA pull-down assay was performed (b1) and the associated proteins were detected with anti-EGFR antibody (b2). A schematic map of potential EGFR binding regions (R1 to 3) in lnc-EGFR (b3). Triangles indicate deletion mutations. ( c ) Lnc-EGFR specifically interacts with EGFR. c1: RIP assays were performed using CD4 + T cells transduced with either lnc-EGFR or lnc-EGFRΔR1 lentiviral particles, and anti-EGFR or anti-PDGFR antibodies. The precipitated RNAs were determined by qPCR for lnc-EGFR, lnc-EGFRΔR1, GAPDH or U6. c2: CD4 + T cells were transduced with different doses of lnc-EGFR lentiviral particles and the association of lnc-EGFR with EGFR was determined by RIP assay using anti-EGFR antibodies and qPCR for lnc-EGFR. c3: The amplified sequence (Lnc-EGFR range from 337 to 379 bp) was validated by Sanger sequencing. Each experiment was performed triplicated. Cytoplasmic lnc-EGFR bind specifically to EGFR. ( a ) Lnc-EGFR RNA pull-down assay was performed. The associated proteins were processed and subjected to Mass Spec. followed by analysis via the Proteome Discoverer program (upper) and the NCBI protein database with the Mascot search engine (lower). ( b ) RNA pull-down assay was performed (upper) and the associated proteins were detected with anti-EGFR antibody (lower panel). ( c ) A schematic map of potential EGFR binding regions (R1 to 3) in lnc-EGFR. Triangles indicate deletion mutations. ( d ) Lnc-EGFR specifically interacts with EGFR. RIP assays were performed using CD4 + T cells transduced with either lnc-EGFR or lnc-EGFRΔR1 lentiviral particles, and anti-EGFR or anti-PDGFR antibodies. The precipitated RNAs were determined by qPCR for lnc-EGFR, lnc-EGFRΔR1, GAPDH or U6. ( e ) CD4 + T cells were transduced with different doses of lnc-EGFR lentiviral particles and the association of lnc-EGFR with EGFR was determined by RIP assay using anti-EGFR antibodies and qPCR for lnc-EGFR. ( f ) The amplified sequence (Lnc-EGFR range from 337 to 379 bp) was validated by Sanger sequencing. Each experiment was performed triplicated.
    Figure Legend Snippet: Cytoplasmic lnc-EGFR binds specifically to EGFR. ( a ) Lnc-EGFR RNA pull-down assay was performed. The associated proteins were processed and subjected to Mass Spec. followed by analysis via the Proteome Discoverer program (a1) and the NCBI protein database with the Mascot search engine (a2). ( b ) RNA pull-down assay was performed (b1) and the associated proteins were detected with anti-EGFR antibody (b2). A schematic map of potential EGFR binding regions (R1 to 3) in lnc-EGFR (b3). Triangles indicate deletion mutations. ( c ) Lnc-EGFR specifically interacts with EGFR. c1: RIP assays were performed using CD4 + T cells transduced with either lnc-EGFR or lnc-EGFRΔR1 lentiviral particles, and anti-EGFR or anti-PDGFR antibodies. The precipitated RNAs were determined by qPCR for lnc-EGFR, lnc-EGFRΔR1, GAPDH or U6. c2: CD4 + T cells were transduced with different doses of lnc-EGFR lentiviral particles and the association of lnc-EGFR with EGFR was determined by RIP assay using anti-EGFR antibodies and qPCR for lnc-EGFR. c3: The amplified sequence (Lnc-EGFR range from 337 to 379 bp) was validated by Sanger sequencing. Each experiment was performed triplicated. Cytoplasmic lnc-EGFR bind specifically to EGFR. ( a ) Lnc-EGFR RNA pull-down assay was performed. The associated proteins were processed and subjected to Mass Spec. followed by analysis via the Proteome Discoverer program (upper) and the NCBI protein database with the Mascot search engine (lower). ( b ) RNA pull-down assay was performed (upper) and the associated proteins were detected with anti-EGFR antibody (lower panel). ( c ) A schematic map of potential EGFR binding regions (R1 to 3) in lnc-EGFR. Triangles indicate deletion mutations. ( d ) Lnc-EGFR specifically interacts with EGFR. RIP assays were performed using CD4 + T cells transduced with either lnc-EGFR or lnc-EGFRΔR1 lentiviral particles, and anti-EGFR or anti-PDGFR antibodies. The precipitated RNAs were determined by qPCR for lnc-EGFR, lnc-EGFRΔR1, GAPDH or U6. ( e ) CD4 + T cells were transduced with different doses of lnc-EGFR lentiviral particles and the association of lnc-EGFR with EGFR was determined by RIP assay using anti-EGFR antibodies and qPCR for lnc-EGFR. ( f ) The amplified sequence (Lnc-EGFR range from 337 to 379 bp) was validated by Sanger sequencing. Each experiment was performed triplicated.

    Techniques Used: Pull Down Assay, Mass Spectrometry, Binding Assay, Transduction, Real-time Polymerase Chain Reaction, Amplification, Sequencing

    15) Product Images from "Dependence on the MUC1-C Oncoprotein in Non-Small Cell Lung Cancer Cells"

    Article Title: Dependence on the MUC1-C Oncoprotein in Non-Small Cell Lung Cancer Cells

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-10-1050

    EGFR mutant NSCLC cells are sensitive to treatment with MUC1-C inhibitors
    Figure Legend Snippet: EGFR mutant NSCLC cells are sensitive to treatment with MUC1-C inhibitors

    Techniques Used: Mutagenesis

    16) Product Images from "Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model"

    Article Title: Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model

    Journal: European Journal of Nuclear Medicine and Molecular Imaging

    doi: 10.1007/s00259-015-3260-x

    Expression of EGFR and CD44v6 and binding specificity in a A431 and b UM-SCC-74B cells using radioimmunoanalysis. Cells were exposed to 0.01 to 60 nM of 124 I/ 125 I-cetuximab or 124 I/ 125 I-AbD19384 and a 100-fold excess of unlabelled antibody was added at the highest concentrations to correct for nonspecific binding. The cells were counted and radioactivity measured in a gamma counter ( n = 3, error bars SD)
    Figure Legend Snippet: Expression of EGFR and CD44v6 and binding specificity in a A431 and b UM-SCC-74B cells using radioimmunoanalysis. Cells were exposed to 0.01 to 60 nM of 124 I/ 125 I-cetuximab or 124 I/ 125 I-AbD19384 and a 100-fold excess of unlabelled antibody was added at the highest concentrations to correct for nonspecific binding. The cells were counted and radioactivity measured in a gamma counter ( n = 3, error bars SD)

    Techniques Used: Expressing, Binding Assay, Radioactivity

    Tumour uptake of a 124 I-Cetuximab ( n = 6, 48 h after injection), b 124 I-AbD19384 ( n = 6, 48 h after injection) and c 18 F-FDG ( n = 7, 1 h after injection) in A431 tumours with high EGFR/CD44v6 expression and in UM-SCC-74B tumours with low EGFR/CD44v6 expression in control and AT13387-treated animals
    Figure Legend Snippet: Tumour uptake of a 124 I-Cetuximab ( n = 6, 48 h after injection), b 124 I-AbD19384 ( n = 6, 48 h after injection) and c 18 F-FDG ( n = 7, 1 h after injection) in A431 tumours with high EGFR/CD44v6 expression and in UM-SCC-74B tumours with low EGFR/CD44v6 expression in control and AT13387-treated animals

    Techniques Used: Injection, Expressing

    Representative small-animal PET/CT images of A431 tumours with high EGFR and CD44v6 expression ( T 1 left posterior flank) and UM-SCC-74B tumours with low EGFR and CD44v6 expression ( T 2 right posterior flank) in nude mice after intravenous injection of a 124 I-cetuximab, b 124 I-AbD19384, and c 18 F-FDG. The upper row shows representative cross sections of the xenografts. The lower row shows planar maximum intensity projection images of the tracer distribution
    Figure Legend Snippet: Representative small-animal PET/CT images of A431 tumours with high EGFR and CD44v6 expression ( T 1 left posterior flank) and UM-SCC-74B tumours with low EGFR and CD44v6 expression ( T 2 right posterior flank) in nude mice after intravenous injection of a 124 I-cetuximab, b 124 I-AbD19384, and c 18 F-FDG. The upper row shows representative cross sections of the xenografts. The lower row shows planar maximum intensity projection images of the tracer distribution

    Techniques Used: Positron Emission Tomography, Expressing, Mouse Assay, Injection

    a c Ex vivo immunohistochemical staining for HSP90, EGFR and CD44v6 expression on representative sections of a A431 and c UM-SCC-74B tumour xenografts (×10). A431 tumours show high expression and UM-SCC-74B tumours show low expression of EGFR and CD44v6. HSP90 and EGFR were downregulated in the AT13387 treatment group. CD44v6 expression was unchanged. b , d Semiquantitative analysis of immunostaining using the H-score ( n = 16, error bars SD; **** p
    Figure Legend Snippet: a c Ex vivo immunohistochemical staining for HSP90, EGFR and CD44v6 expression on representative sections of a A431 and c UM-SCC-74B tumour xenografts (×10). A431 tumours show high expression and UM-SCC-74B tumours show low expression of EGFR and CD44v6. HSP90 and EGFR were downregulated in the AT13387 treatment group. CD44v6 expression was unchanged. b , d Semiquantitative analysis of immunostaining using the H-score ( n = 16, error bars SD; **** p

    Techniques Used: Ex Vivo, Immunohistochemistry, Staining, Expressing, Immunostaining

    17) Product Images from "Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer"

    Article Title: Honeybee venom and melittin suppress growth factor receptor activation in HER2-enriched and triple-negative breast cancer

    Journal: NPJ Precision Oncology

    doi: 10.1038/s41698-020-00129-0

    Honeybee venom and melittin suppress the phosphorylation of EGFR and HER2. a Phosphorylation kinetics of HER2, EGFR, and downstream MAPK and Akt pathways after treatment with honeybee venom and melittin in SKBR3 (left) and SUM159 (right) breast cancer cells, assessed by immunoblotting. b Bioluminescence resonance energy transfer (BRET) kinetic analysis of TAMRA-EGF, FITC-melittin, and FITC–DEDE-melittin interaction with NanoLuc-EGFR in HEK293FT cells. The peptides were added after the cells were equilibrated in the reader with the NanoLuc substrate furimazine for 5 min. c Saturation-binding analysis of increasing concentrations of TAMRA-EGF, FITC-melittin, and FITC–DEDE-melittin in HEK293FT cells transfected with NanoLuc-EGFR in the presence or absence of unlabeled EGF (1 µM). Data are expressed as raw BRET ratios and represented as mean ± SEM ( n = 3, two-way ANOVA). d Proposed model of action of melittin interfering with the dimerization and phosphorylation of RTKs in the plasma membrane. Differences were considered significant at p
    Figure Legend Snippet: Honeybee venom and melittin suppress the phosphorylation of EGFR and HER2. a Phosphorylation kinetics of HER2, EGFR, and downstream MAPK and Akt pathways after treatment with honeybee venom and melittin in SKBR3 (left) and SUM159 (right) breast cancer cells, assessed by immunoblotting. b Bioluminescence resonance energy transfer (BRET) kinetic analysis of TAMRA-EGF, FITC-melittin, and FITC–DEDE-melittin interaction with NanoLuc-EGFR in HEK293FT cells. The peptides were added after the cells were equilibrated in the reader with the NanoLuc substrate furimazine for 5 min. c Saturation-binding analysis of increasing concentrations of TAMRA-EGF, FITC-melittin, and FITC–DEDE-melittin in HEK293FT cells transfected with NanoLuc-EGFR in the presence or absence of unlabeled EGF (1 µM). Data are expressed as raw BRET ratios and represented as mean ± SEM ( n = 3, two-way ANOVA). d Proposed model of action of melittin interfering with the dimerization and phosphorylation of RTKs in the plasma membrane. Differences were considered significant at p

    Techniques Used: Bioluminescence Resonance Energy Transfer, Binding Assay, Transfection

    Engineering melittin with an RGD motif enhances breast cancer selectivity. a Cell-viability assays of TNBC (SUM159) and HER2-enriched breast cancer (SKBR3) cells treated with DEDE-melittin for 24 h. b Cell-viability assays of T11 cells treated with melittin, RGD1-melittin, SV40-melittin, and DEDE-melittin for 24 h ( t test). c Cell-viability assays of normal human dermal fibroblasts (HDFa) and SUM159 treated with melittin (left) and RGD1-melittin (right) for 24 h ( t tests). d Western blot for the detection of cleaved caspase-3 (CL-csp-3) in lysates from SUM159 cells treated with vehicle, melittin, DEDE-melittin, or RGD1-melittin for 24 h. e Absorbance (405 nm) of aqueous solutions of melittin, RGD1-melittin, DEDE-melittin, and SV40-melittin subjected to an ELISA with the anti-melittin antibody (two-way ANOVA). f The amino-acid sequence and top predicted 3D model of melittin (green), RGD1-melittin (purple), DEDE-melittin (blue), and SV40-melittin (orange). g Immunofluorescence images of SUM159 treated with vehicle, honeybee venom, melittin, RGD1-melittin, or DEDE-melittin for 30 min. In blue: cell nuclei, in red: anti-EGFR, and in green: anti-melittin. The white outlines in the merged images indicate the respective regions in the zoomed images. Scale bars represent 25 µm, and 6.25 µm for the zoomed images. Data are represented as mean ± SEM ( n = 3). Differences were considered significant at p
    Figure Legend Snippet: Engineering melittin with an RGD motif enhances breast cancer selectivity. a Cell-viability assays of TNBC (SUM159) and HER2-enriched breast cancer (SKBR3) cells treated with DEDE-melittin for 24 h. b Cell-viability assays of T11 cells treated with melittin, RGD1-melittin, SV40-melittin, and DEDE-melittin for 24 h ( t test). c Cell-viability assays of normal human dermal fibroblasts (HDFa) and SUM159 treated with melittin (left) and RGD1-melittin (right) for 24 h ( t tests). d Western blot for the detection of cleaved caspase-3 (CL-csp-3) in lysates from SUM159 cells treated with vehicle, melittin, DEDE-melittin, or RGD1-melittin for 24 h. e Absorbance (405 nm) of aqueous solutions of melittin, RGD1-melittin, DEDE-melittin, and SV40-melittin subjected to an ELISA with the anti-melittin antibody (two-way ANOVA). f The amino-acid sequence and top predicted 3D model of melittin (green), RGD1-melittin (purple), DEDE-melittin (blue), and SV40-melittin (orange). g Immunofluorescence images of SUM159 treated with vehicle, honeybee venom, melittin, RGD1-melittin, or DEDE-melittin for 30 min. In blue: cell nuclei, in red: anti-EGFR, and in green: anti-melittin. The white outlines in the merged images indicate the respective regions in the zoomed images. Scale bars represent 25 µm, and 6.25 µm for the zoomed images. Data are represented as mean ± SEM ( n = 3). Differences were considered significant at p

    Techniques Used: Western Blot, Enzyme-linked Immunosorbent Assay, Sequencing, Immunofluorescence

    18) Product Images from "The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells"

    Article Title: The novel HSP90 inhibitor AT13387 potentiates radiation effects in squamous cell carcinoma and adenocarcinoma cells

    Journal: Oncotarget

    doi:

    Effect of AT13387 treatment on HSP90 client protein levels (HSP90, ATM, DNA-PKcs, EGFR, AKT, CD44, CD44v6) A. Dose-dependent downregulation of client proteins. H314 cells and LS1474T cells treated with the indicated doses of AT13387 for 24 h. B. Recurrence of protein level after AT13387 treatment. H314 and LS174T cells were treated with 200 nM AT13387 for 24 h. After drug treatment cells were kept in drug-free complete medium for 0, 4, 8 and 24 h. Lysates were harvested and equivalent amounts of protein from each lysate were resolved by SDS-PAGE and immunoblotting with the indicated antibodies. The expression levels of beta actin were used to ensure equal loading. Above: Representative Western blots, Below: Western blot quantification. Protein levels were normalized to beta actin, and were normalized to the level of untreated control (dashed line). One way-ANOVA with Bonferroni post-test was used to calculate statistics: * p
    Figure Legend Snippet: Effect of AT13387 treatment on HSP90 client protein levels (HSP90, ATM, DNA-PKcs, EGFR, AKT, CD44, CD44v6) A. Dose-dependent downregulation of client proteins. H314 cells and LS1474T cells treated with the indicated doses of AT13387 for 24 h. B. Recurrence of protein level after AT13387 treatment. H314 and LS174T cells were treated with 200 nM AT13387 for 24 h. After drug treatment cells were kept in drug-free complete medium for 0, 4, 8 and 24 h. Lysates were harvested and equivalent amounts of protein from each lysate were resolved by SDS-PAGE and immunoblotting with the indicated antibodies. The expression levels of beta actin were used to ensure equal loading. Above: Representative Western blots, Below: Western blot quantification. Protein levels were normalized to beta actin, and were normalized to the level of untreated control (dashed line). One way-ANOVA with Bonferroni post-test was used to calculate statistics: * p

    Techniques Used: SDS Page, Expressing, Western Blot

    Immunohistochemical analysis on A431 tumors A. Representative images of ex vivo immunohistochemical staining for HSP90, EGFR, CD44, CD44v6, DNA-PKcs, ATM and MET expression in A431 tumor xenografts (magnification × 10). Mice in the treatment group ( n = 6) received 5 doses of 50 mg/kg AT13387 on 5 consecutive days before dissection and analysis. The effect of AT13387 was highest on the expression pattern of HSP90, EGFR and MET. B. Semiquantitative analysis of immunostainings using the H-score method. Error bars represent the standard deviation, n = 16. One way-ANOVA with Bonferroni post-test was used to calculate statistics: * p
    Figure Legend Snippet: Immunohistochemical analysis on A431 tumors A. Representative images of ex vivo immunohistochemical staining for HSP90, EGFR, CD44, CD44v6, DNA-PKcs, ATM and MET expression in A431 tumor xenografts (magnification × 10). Mice in the treatment group ( n = 6) received 5 doses of 50 mg/kg AT13387 on 5 consecutive days before dissection and analysis. The effect of AT13387 was highest on the expression pattern of HSP90, EGFR and MET. B. Semiquantitative analysis of immunostainings using the H-score method. Error bars represent the standard deviation, n = 16. One way-ANOVA with Bonferroni post-test was used to calculate statistics: * p

    Techniques Used: Immunohistochemistry, Ex Vivo, Staining, Expressing, Mouse Assay, Dissection, Standard Deviation

    19) Product Images from "Identification of a novel subpopulation of Caspase-4 positive non-small cell lung Cancer patients"

    Article Title: Identification of a novel subpopulation of Caspase-4 positive non-small cell lung Cancer patients

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-020-01754-0

    Large subunit of caspase-4 facilitates tumor cell proliferation. A549 cells were transfected with PC4–1, PC4–2, PC4–3 and PC4–4 as explained in Materials and Method section. a Cells transfected with PC4–4 (the sole large subunit of caspase-4) showed higher CFSE positive cells than the other groups ( n = 15). b Co-culture of A549 cells and PBMCs (ratio 1:5) showed similar proliferation expressed as %CFSE+ cells than A549 alone treated with the recombinant large subunit of capase-4. The neutralization of EGFR by means of cetuximab ( c ), inhibition of HDAC by means of SAHA ( d ), inhibition of methyltransferase ( e ) and mTOR ( g ) did not alter cell proliferation as instead observed when the inhibitor of k-RAS (FTI-276) was added ( f ). Experiments were performed n = 15 in duplicate. Data are expressed as median ± quartile range and represented as violin plots. One-Way ANOVA followed by Bonferroni’s post test was applied
    Figure Legend Snippet: Large subunit of caspase-4 facilitates tumor cell proliferation. A549 cells were transfected with PC4–1, PC4–2, PC4–3 and PC4–4 as explained in Materials and Method section. a Cells transfected with PC4–4 (the sole large subunit of caspase-4) showed higher CFSE positive cells than the other groups ( n = 15). b Co-culture of A549 cells and PBMCs (ratio 1:5) showed similar proliferation expressed as %CFSE+ cells than A549 alone treated with the recombinant large subunit of capase-4. The neutralization of EGFR by means of cetuximab ( c ), inhibition of HDAC by means of SAHA ( d ), inhibition of methyltransferase ( e ) and mTOR ( g ) did not alter cell proliferation as instead observed when the inhibitor of k-RAS (FTI-276) was added ( f ). Experiments were performed n = 15 in duplicate. Data are expressed as median ± quartile range and represented as violin plots. One-Way ANOVA followed by Bonferroni’s post test was applied

    Techniques Used: Transfection, Co-Culture Assay, Recombinant, Neutralization, Inhibition

    20) Product Images from "Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways *"

    Article Title: Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Epiphyseal Cartilage Development through β-Catenin-dependent and -independent Pathways *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.463554

    The development of SOC in the proximal epiphysis of the tibia is delayed in the chondrocyte-specific EGFR-deficient mice. A , immunohistochemistry of EGFR in the epiphyseal cartilage of P9 WT mice reveals that most chondrocytes express EGFR ( a ). Black
    Figure Legend Snippet: The development of SOC in the proximal epiphysis of the tibia is delayed in the chondrocyte-specific EGFR-deficient mice. A , immunohistochemistry of EGFR in the epiphyseal cartilage of P9 WT mice reveals that most chondrocytes express EGFR ( a ). Black

    Techniques Used: Mouse Assay, Immunohistochemistry

    EGFR signaling stimulates the expression of Mmp9 and -13 and Rankl through β-catenin-dependent and -independent pathways. A , TGFα stimulates the expression of Mmp9 and -13 and Rankl in mouse chondrocytes through the EGFR pathway. Mouse
    Figure Legend Snippet: EGFR signaling stimulates the expression of Mmp9 and -13 and Rankl through β-catenin-dependent and -independent pathways. A , TGFα stimulates the expression of Mmp9 and -13 and Rankl in mouse chondrocytes through the EGFR pathway. Mouse

    Techniques Used: Expressing

    EGFR deficiency in chondrocytes suppresses the terminal differentiation and apoptosis of hypertrophic chondrocytes adjacent to the marrow space in the SOC. A , immunohistochemistry of Runx2 protein ( a–c ) and TUNEL ( d–f ) staining in sections
    Figure Legend Snippet: EGFR deficiency in chondrocytes suppresses the terminal differentiation and apoptosis of hypertrophic chondrocytes adjacent to the marrow space in the SOC. A , immunohistochemistry of Runx2 protein ( a–c ) and TUNEL ( d–f ) staining in sections

    Techniques Used: Immunohistochemistry, TUNEL Assay, Staining

    Blocking EGFR activity in chondrocytes reduces the amounts of MMPs and cartilage matrix degradation in the hypertrophic chondrocytes adjacent to the marrow space in the SOC. A , immunohistochemistry of MMP9 ( a–c ), 13 ( d–f ), 14 ( g–i
    Figure Legend Snippet: Blocking EGFR activity in chondrocytes reduces the amounts of MMPs and cartilage matrix degradation in the hypertrophic chondrocytes adjacent to the marrow space in the SOC. A , immunohistochemistry of MMP9 ( a–c ), 13 ( d–f ), 14 ( g–i

    Techniques Used: Blocking Assay, Activity Assay, Immunohistochemistry

    21) Product Images from "DaHuangWan targets EGF signaling to inhibit the proliferation of hepatoma cells"

    Article Title: DaHuangWan targets EGF signaling to inhibit the proliferation of hepatoma cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0231466

    A, PLC/PFR/5 cells were serum-starved and incubated without (control) or with Berberine, Costunolide and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins. The expression level of P-EGFR in PLC/PRF/5 cells was examined by Western blot. B, EGFR on the surface of PLC/PRF/5 cells were examined by immunofluorescence after treated with Berberine, Costunolide and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins; C, PLC/PFR/5 cells were serum-starved and incubated without (control) or with Berberine, Costunolide, and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins. Proteins on cell surface were labeled with Sulfo-NHS-SS-Biotin and isolated by streptavidin and then analyzed by immunoblot detected EGFR (lane5-8); The internalized cell surface proteins were isolated using Sulfo-NHS-SS-Biotin and MesNa and analyzed by immunoblot detecting EGFR (top panel) (lane9-12). D, PLC/PRF/5 cells were incubated without (control) or with Berberine, Costunolide, and BC mix for the indicated time periods. Ubiquitinated EGFR was examined via EGFR immunoprecipitation followed by immunoblotting with an ubiquitin antibody. The bands were quantified. The level of ubiquitinated EGFR was normalized to the immunoprecipitated EGFR. The relative density of the control.
    Figure Legend Snippet: A, PLC/PFR/5 cells were serum-starved and incubated without (control) or with Berberine, Costunolide and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins. The expression level of P-EGFR in PLC/PRF/5 cells was examined by Western blot. B, EGFR on the surface of PLC/PRF/5 cells were examined by immunofluorescence after treated with Berberine, Costunolide and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins; C, PLC/PFR/5 cells were serum-starved and incubated without (control) or with Berberine, Costunolide, and BC mix for 24 hrs, and then stimulated by EGF (10ng/ml) for 30 mins. Proteins on cell surface were labeled with Sulfo-NHS-SS-Biotin and isolated by streptavidin and then analyzed by immunoblot detected EGFR (lane5-8); The internalized cell surface proteins were isolated using Sulfo-NHS-SS-Biotin and MesNa and analyzed by immunoblot detecting EGFR (top panel) (lane9-12). D, PLC/PRF/5 cells were incubated without (control) or with Berberine, Costunolide, and BC mix for the indicated time periods. Ubiquitinated EGFR was examined via EGFR immunoprecipitation followed by immunoblotting with an ubiquitin antibody. The bands were quantified. The level of ubiquitinated EGFR was normalized to the immunoprecipitated EGFR. The relative density of the control.

    Techniques Used: Planar Chromatography, Incubation, Expressing, Western Blot, Immunofluorescence, Labeling, Isolation, Immunoprecipitation

    22) Product Images from "The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation"

    Article Title: The MARCH Family E3 Ubiquitin Ligase K5 Alters Monocyte Metabolism and Proliferation through Receptor Tyrosine Kinase Modulation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001331

    K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.
    Figure Legend Snippet: K5 interacts with and alters RTK localization. Equal cell numbers of the indicated THP-1 lines were fixed with paraformaldehyde (PFA) and ( A ) stained without permeabilization to determine surface expression or ( B ) permeabilized with saponin prior to staining to determine total expression of Flt-4, PDGFR-ß, Flt-3 and EGFR by flow cytometry. Data are representative of three independent experiments. ( B, Inset ) The relative ratio of surface versus total RTKs was determined for vector- and K5 WT-expressing THP-1 cells. ( C ) 293T cells were co-transfected with expression constructs for EGFR, PDGFR-ß, or Flt-4 and the indicated GST expression constructs. After two days, lysates were subjected to GST pull-down using glutathione-sepharose beads. Purified proteins and whole cell lysates (WCL) were subjected to western blot (WB) using anti-EGFR, -Flt-4 or -PDGFR-ß antibodies, followed by re-probing with anti-GST antibodies. Arrows indicate GST or GST-K5 WT and mutant specific bands. Data are representative of three independent experiments.

    Techniques Used: Staining, Expressing, Flow Cytometry, Cytometry, Plasmid Preparation, Transfection, Construct, Purification, Western Blot, Mutagenesis

    23) Product Images from "ErbB3 Ligand Heregulin1 Is a Major Mitogenic Factor for Uncontrolled Lung Cancer Cell Proliferation"

    Article Title: ErbB3 Ligand Heregulin1 Is a Major Mitogenic Factor for Uncontrolled Lung Cancer Cell Proliferation

    Journal: Neoplasia (New York, N.Y.)

    doi: 10.1016/j.neo.2019.02.001

    Comparison of expression level of EGFR and ErbB3 ligands on NSCLC samples and cell lines. (A) IHC was performed by following standard procedures of hospital. Expression level of ligands between tumor and nontumor cells is determined by TisssueGnostics StrataQuest System. (B): Real-time PCR was performed with 200 ng of RNA with an iTaq Universal SYBR One-Step Kit. (A-F) Fold expression was calculated as relative fold expression = 2 cq value of GAPDH-cq value of receptors . Data are plotted as mean ± SD of triplicates. (B-F) (C) Amplification efficiency of primers: RFUs from eight amplification cycles post threshold were used to compare the amplification efficiency for each pair of primers. Triplicates were set up for each pairs of primer. The data are representative of two independent experiments.
    Figure Legend Snippet: Comparison of expression level of EGFR and ErbB3 ligands on NSCLC samples and cell lines. (A) IHC was performed by following standard procedures of hospital. Expression level of ligands between tumor and nontumor cells is determined by TisssueGnostics StrataQuest System. (B): Real-time PCR was performed with 200 ng of RNA with an iTaq Universal SYBR One-Step Kit. (A-F) Fold expression was calculated as relative fold expression = 2 cq value of GAPDH-cq value of receptors . Data are plotted as mean ± SD of triplicates. (B-F) (C) Amplification efficiency of primers: RFUs from eight amplification cycles post threshold were used to compare the amplification efficiency for each pair of primers. Triplicates were set up for each pairs of primer. The data are representative of two independent experiments.

    Techniques Used: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Amplification

    EGF, TGF-a, BTC, and HB-EGF are inhibitory factors on HRG1-induced proliferation of cells with EGFRwt-ErbB3 but not with EGFRmut-ErbB3: (A-D) EGFR-ErbB3–expressing FDC-P1cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands at 20 ng/ml at different times. (E-F) NSCLC cells were seeded in 96-well plates and were treated with EGFR and ErbB3 ligands at 20 ng/ml for 72 hours. The relative cell number was determined with CCK8. The OD value at 450 nm was measured with an iMark microplate reader. Results were shown as cell growth curves for EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing FDC-P1 cells in response to different combinations of EGFR and ErbB3 ligand treatment respectively (A-D). The difference above the nontreatment control was compared to show the effect of HRG1 vs. HRG1 plus EGFR ligand treatment for NSCLC cells (E-F). Data are plotted as mean ± SD of tetraplicates. The data are representative of three independent experiments.
    Figure Legend Snippet: EGF, TGF-a, BTC, and HB-EGF are inhibitory factors on HRG1-induced proliferation of cells with EGFRwt-ErbB3 but not with EGFRmut-ErbB3: (A-D) EGFR-ErbB3–expressing FDC-P1cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands at 20 ng/ml at different times. (E-F) NSCLC cells were seeded in 96-well plates and were treated with EGFR and ErbB3 ligands at 20 ng/ml for 72 hours. The relative cell number was determined with CCK8. The OD value at 450 nm was measured with an iMark microplate reader. Results were shown as cell growth curves for EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing FDC-P1 cells in response to different combinations of EGFR and ErbB3 ligand treatment respectively (A-D). The difference above the nontreatment control was compared to show the effect of HRG1 vs. HRG1 plus EGFR ligand treatment for NSCLC cells (E-F). Data are plotted as mean ± SD of tetraplicates. The data are representative of three independent experiments.

    Techniques Used: Expressing

    (A) HRG1 and 2 preferably activate EGFRmut-ErbB3 to EGFRwt-ErbB3 heterodimer. (B) Heterodimer is required for HRG1 and 2 to function in FDC-P1 cells. EGFR-ErbB3-expressing FDC-P1 cells and EGFR- or ErbB3-expressing FDC-P1 single receptor cells were seeded in 96-well plates and treated with different ligands at 20 ng/ml for different times. The relative cell number was determined with CCK8. OD value at 450 nm was measured with an iMark microplate reader. Data are plotted as mean ± SD of tetraplicates. (C) Effect of different EGFR ligands on EGFRwt-ErbB3 and EGFRmut-ErbB3 cell proliferation. Cells were seed at 8×10 4 /ml in 6-well plates; cell numbers were counted each day. Data are plotted as mean± SD of tetraplicates. The data are representative of three independent experiments.
    Figure Legend Snippet: (A) HRG1 and 2 preferably activate EGFRmut-ErbB3 to EGFRwt-ErbB3 heterodimer. (B) Heterodimer is required for HRG1 and 2 to function in FDC-P1 cells. EGFR-ErbB3-expressing FDC-P1 cells and EGFR- or ErbB3-expressing FDC-P1 single receptor cells were seeded in 96-well plates and treated with different ligands at 20 ng/ml for different times. The relative cell number was determined with CCK8. OD value at 450 nm was measured with an iMark microplate reader. Data are plotted as mean ± SD of tetraplicates. (C) Effect of different EGFR ligands on EGFRwt-ErbB3 and EGFRmut-ErbB3 cell proliferation. Cells were seed at 8×10 4 /ml in 6-well plates; cell numbers were counted each day. Data are plotted as mean± SD of tetraplicates. The data are representative of three independent experiments.

    Techniques Used: Expressing

    Comparison of expression level of EGFR and ErbB3 on EGFR-ErbB3 expressing FDC-P1 cells and NSCLC cell lines. Real-time PCR was performed with 200 ng of RNA with an iTaq Universal SYBR One-Step Kit. Fold expression was calculated as relative fold expression = 2 cq value of GAPDH-cq value of receptors . Data are plotted as mean ± SD of triplicates.
    Figure Legend Snippet: Comparison of expression level of EGFR and ErbB3 on EGFR-ErbB3 expressing FDC-P1 cells and NSCLC cell lines. Real-time PCR was performed with 200 ng of RNA with an iTaq Universal SYBR One-Step Kit. Fold expression was calculated as relative fold expression = 2 cq value of GAPDH-cq value of receptors . Data are plotted as mean ± SD of triplicates.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    (A) High expression of EGFR, ErbB3, and their ligands: IHC was performed by the following standard procedures of hospital: Pictures were taken at junction area between tumor cells at right side and normal alveoli or stroma tissue at left side with 10×10 amplification from the same one patient with NSCLC harboring EGFR mutant of E746-A750del. (B). Different response to binding of EGFR and ErbB3 ligands: When EGF binds to wild-type or mutant EGFR-ErbB3 heterodimer, the EGFR configuration change allows it to activate MAP kinase pathway and a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (A and E). When HRG1 binds to EGFR-ErbB3 heterodimer, the EGFR configuration change does not allow it to activate MAP kinase pathway but allows it to phosphorylate ErbB3 and generates a strong proliferation signal (B and F). When EGF and HRG1 bind to their receptors simultaneously, the configuration change of wild-type EGFR allows it to activate MAP kinase pathway and activates a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (C), whereas the same event allows mutant EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which results in a stronger proliferation signal (G). When AREG and HRG1 bind to their receptors simultaneously, the configuration change of either wild-type or mutant EGFR allows EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which also results in a stronger proliferation signal (D and H).
    Figure Legend Snippet: (A) High expression of EGFR, ErbB3, and their ligands: IHC was performed by the following standard procedures of hospital: Pictures were taken at junction area between tumor cells at right side and normal alveoli or stroma tissue at left side with 10×10 amplification from the same one patient with NSCLC harboring EGFR mutant of E746-A750del. (B). Different response to binding of EGFR and ErbB3 ligands: When EGF binds to wild-type or mutant EGFR-ErbB3 heterodimer, the EGFR configuration change allows it to activate MAP kinase pathway and a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (A and E). When HRG1 binds to EGFR-ErbB3 heterodimer, the EGFR configuration change does not allow it to activate MAP kinase pathway but allows it to phosphorylate ErbB3 and generates a strong proliferation signal (B and F). When EGF and HRG1 bind to their receptors simultaneously, the configuration change of wild-type EGFR allows it to activate MAP kinase pathway and activates a phosphatase that dephosphorylates ErbB3, which results in a weak proliferation signal (C), whereas the same event allows mutant EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which results in a stronger proliferation signal (G). When AREG and HRG1 bind to their receptors simultaneously, the configuration change of either wild-type or mutant EGFR allows EGFR to activate MAP kinase pathway and phosphorylate ErbB3, which also results in a stronger proliferation signal (D and H).

    Techniques Used: Expressing, Immunohistochemistry, Amplification, Mutagenesis, Binding Assay

    Expression of EGFR, ErbB3, and their ligands on patient samples: 16 tumor tissues were fixed in 4% polyoxymethylene, embedded in paraffin, sectioned to 3 μm, and mounted on adhesion microscope slides. IHC was performed by following standard procedures of hospital. (A) EGFR; (B) ErbB3; (C) HRG1; (D) AREG; (E) EREG.
    Figure Legend Snippet: Expression of EGFR, ErbB3, and their ligands on patient samples: 16 tumor tissues were fixed in 4% polyoxymethylene, embedded in paraffin, sectioned to 3 μm, and mounted on adhesion microscope slides. IHC was performed by following standard procedures of hospital. (A) EGFR; (B) ErbB3; (C) HRG1; (D) AREG; (E) EREG.

    Techniques Used: Expressing, Microscopy, Immunohistochemistry

    Effect of HRG1 and EGFR ligands on EGFR-ErbB3 heterodimer: (A-E) Imaging analysis of EGFR-ErbB3 heterodimer: Cells were seeded on glass-bottom culture dishes. EGF, HRG1, and HRG1 plus EGF at 20 ng/ml were added to cells for 15 minutes. Cells were immunofluorescence stained. STORM imaging was performed with an N-STORM system built on a Nikon-Ti-E inverted microscope with an HP Apo 100× TIRF objective having a numerical aperture of 1.49. Images demonstrate preexisting EGFR-ErbB3 heterodimers. EGF treatment induced ErbB3 internalization, and HRG1 treatment induced EGFR internalization, respectively. (B) 3D STORM images of EGFR-ErbB3 heterodimer were reconstructed using Nikon NIS Elements image analysis software to reflect the level of protein colocalization. Photos of an EGFR-ErbB3 heterodimer were taken from three different directions. The green color represents ErbB3, and the red color represents EGFR. (F) Co-immunoprecipitation: Cells at 50% confluence were serum starved for 24 hours. The EGFR-ErbB3–expressing FDC-P1 cells were untreated or treated with EGF, HRG1, and EGF plus HRG1 at 20 ng/ml for 15 minutes. The cells were lysed in NP-40 buffer with phosphotase inhibitors. EGFR and ErbB3 were immunoprecipitated and subjected to SDS-PAGE. The membranes were probed with anti-ErbB3 or anti-EGFR antibody. (G) Western blot: Cells were serum starved for 24 hours and were untreated or treated with different ligands for 15 minutes except the last two columns where cells were first treated with HRG1 for 14 or 10 minutes and then treated with EGF for 1 or 5 minutes, respectively (total 15 minutes). Cells lyses were subjected to SDS-PAGE. Membranes were probed antiphosphorylated ErbB3, stripped, and reprobed with anti-ErbB3 antibody as loading contro,l respectively. The data are representative of three independent experiments.
    Figure Legend Snippet: Effect of HRG1 and EGFR ligands on EGFR-ErbB3 heterodimer: (A-E) Imaging analysis of EGFR-ErbB3 heterodimer: Cells were seeded on glass-bottom culture dishes. EGF, HRG1, and HRG1 plus EGF at 20 ng/ml were added to cells for 15 minutes. Cells were immunofluorescence stained. STORM imaging was performed with an N-STORM system built on a Nikon-Ti-E inverted microscope with an HP Apo 100× TIRF objective having a numerical aperture of 1.49. Images demonstrate preexisting EGFR-ErbB3 heterodimers. EGF treatment induced ErbB3 internalization, and HRG1 treatment induced EGFR internalization, respectively. (B) 3D STORM images of EGFR-ErbB3 heterodimer were reconstructed using Nikon NIS Elements image analysis software to reflect the level of protein colocalization. Photos of an EGFR-ErbB3 heterodimer were taken from three different directions. The green color represents ErbB3, and the red color represents EGFR. (F) Co-immunoprecipitation: Cells at 50% confluence were serum starved for 24 hours. The EGFR-ErbB3–expressing FDC-P1 cells were untreated or treated with EGF, HRG1, and EGF plus HRG1 at 20 ng/ml for 15 minutes. The cells were lysed in NP-40 buffer with phosphotase inhibitors. EGFR and ErbB3 were immunoprecipitated and subjected to SDS-PAGE. The membranes were probed with anti-ErbB3 or anti-EGFR antibody. (G) Western blot: Cells were serum starved for 24 hours and were untreated or treated with different ligands for 15 minutes except the last two columns where cells were first treated with HRG1 for 14 or 10 minutes and then treated with EGF for 1 or 5 minutes, respectively (total 15 minutes). Cells lyses were subjected to SDS-PAGE. Membranes were probed antiphosphorylated ErbB3, stripped, and reprobed with anti-ErbB3 antibody as loading contro,l respectively. The data are representative of three independent experiments.

    Techniques Used: Imaging, Immunofluorescence, Staining, Inverted Microscopy, Software, Immunoprecipitation, Expressing, SDS Page, Western Blot

    Heregulin1 is the major mitogenic factor for cell proliferation: (A-D) EGFR-ErbB3–expressing FDC-P1 cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands or IL-3 at 20 ng/ml at different times. (E-F) NSCLC cells were seeded in 96-well plates and were treated with EGFR and ErbB3 ligands at 20 ng/ml for 72 hours. The relative cell number was determined with CCCK8. The OD value at 450 nm was measured with an iMark microplate reader. Results were shown as cell growth curves for EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing FDC-P1 cells in response to ligand stimulation, respectively (A-D). The difference above the nontreatment control was compared in order to determine which ligand was the major mitogenic factor for NSCLC cells with EGFRmut (E-F). Data are plotted as mean +/− SD of tetraplicates. The data are representative of three independent experiments.
    Figure Legend Snippet: Heregulin1 is the major mitogenic factor for cell proliferation: (A-D) EGFR-ErbB3–expressing FDC-P1 cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands or IL-3 at 20 ng/ml at different times. (E-F) NSCLC cells were seeded in 96-well plates and were treated with EGFR and ErbB3 ligands at 20 ng/ml for 72 hours. The relative cell number was determined with CCCK8. The OD value at 450 nm was measured with an iMark microplate reader. Results were shown as cell growth curves for EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing FDC-P1 cells in response to ligand stimulation, respectively (A-D). The difference above the nontreatment control was compared in order to determine which ligand was the major mitogenic factor for NSCLC cells with EGFRmut (E-F). Data are plotted as mean +/− SD of tetraplicates. The data are representative of three independent experiments.

    Techniques Used: Expressing

    AREG blocks the inhibition of EGF on HRG1 induced cell proliferation: EGFR-ErbB3–expressing cells were seeded in 96-well plates and treated with different concentration of ligands for 72 hours. All concentrations of ligands were 20n g/ml except those that were specifically labeled for EGF. The relative cell number was determined with a CCK8. The OD value at 450 nm was measured to show the effect of the ratio of EGF/AREG on HRG1-induced cell proliferation. Data are plotted as mean ± SD of tetraplicates. The data are representative of three independent experiments.
    Figure Legend Snippet: AREG blocks the inhibition of EGF on HRG1 induced cell proliferation: EGFR-ErbB3–expressing cells were seeded in 96-well plates and treated with different concentration of ligands for 72 hours. All concentrations of ligands were 20n g/ml except those that were specifically labeled for EGF. The relative cell number was determined with a CCK8. The OD value at 450 nm was measured to show the effect of the ratio of EGF/AREG on HRG1-induced cell proliferation. Data are plotted as mean ± SD of tetraplicates. The data are representative of three independent experiments.

    Techniques Used: Inhibition, Expressing, Concentration Assay, Labeling

    Effect of different ligands and different ligand combinations on proliferation of cells with EGFRwt-ErbB3 and EGFRmut-ErbB3: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing bone marrow cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands and different combination of these ligands at different times. The relative cell number was determined with CCK8. The OD value at 450 nm was measured with an iMark microplate reader. Data are plotted as mean ± SD of tetraplicates. (A) Effect of HRG1 on EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. (B) Effect of IL-3 on EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation. (C) Effect of different ligands on EGFRwt-ErbB3–expressing cell proliferation. (D) Effect of different ligands on EGFRmut-ErbB3–expressing cell proliferation. (E-F) Effect of four EGFR ligands on HRG1-induced EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. (G-H) Effect of other three EGFR ligands on HRG1-induced EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. The data are representative of three independent experiments.
    Figure Legend Snippet: Effect of different ligands and different ligand combinations on proliferation of cells with EGFRwt-ErbB3 and EGFRmut-ErbB3: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing bone marrow cells were seeded in 96-well plates and treated with different EGFR and ErbB3 ligands and different combination of these ligands at different times. The relative cell number was determined with CCK8. The OD value at 450 nm was measured with an iMark microplate reader. Data are plotted as mean ± SD of tetraplicates. (A) Effect of HRG1 on EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. (B) Effect of IL-3 on EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation. (C) Effect of different ligands on EGFRwt-ErbB3–expressing cell proliferation. (D) Effect of different ligands on EGFRmut-ErbB3–expressing cell proliferation. (E-F) Effect of four EGFR ligands on HRG1-induced EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. (G-H) Effect of other three EGFR ligands on HRG1-induced EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing cell proliferation, respectively. The data are representative of three independent experiments.

    Techniques Used: Expressing

    EGF, TGF-a, BTC, and HB-EGF activate EGFRwt and EGFRmut equally; AREG, EREG, and EPGN are weak activators for EGFRwt but as potent as other four activators for EGFRmut: (A-B) Western blot assay: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing CHO Cells and lung cancer cells at 50% confluence were serum starved for 24 hours. The cells were untreated or treated with different EGFR ligands at different concentration for 15 minutes. The lyses were subjected to SDS-PAGE. Membranes were probed, stripped, and reprobed with different antibodies. Results showed the effect of different EGFR ligands on EGFRwt and EGFRmut activation. The data are representative of four independent experiments. (C-D) Immunofluorescence: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing CHO cells and lung cancer cells were grown on 14-mm glass coverslips in 24-well plates, serum starved overnight, and treated with different EGFR ligands for 30 minutes. The cells were immunofluorescence stained. Pictures were taken with a 100× oil lens on the Nikon A1 Laser Scanning Confocal Microscope. The data show the effect of different EGFR ligands on the nuclear translocation of EGFRwt and EGFRmut. Pictures were taken with a 100× oil lens on the Nikon A1 laser scanning confocal microscope. The data are representative of three independent experiments.
    Figure Legend Snippet: EGF, TGF-a, BTC, and HB-EGF activate EGFRwt and EGFRmut equally; AREG, EREG, and EPGN are weak activators for EGFRwt but as potent as other four activators for EGFRmut: (A-B) Western blot assay: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing CHO Cells and lung cancer cells at 50% confluence were serum starved for 24 hours. The cells were untreated or treated with different EGFR ligands at different concentration for 15 minutes. The lyses were subjected to SDS-PAGE. Membranes were probed, stripped, and reprobed with different antibodies. Results showed the effect of different EGFR ligands on EGFRwt and EGFRmut activation. The data are representative of four independent experiments. (C-D) Immunofluorescence: EGFRwt-ErbB3– and EGFRmut-ErbB3–expressing CHO cells and lung cancer cells were grown on 14-mm glass coverslips in 24-well plates, serum starved overnight, and treated with different EGFR ligands for 30 minutes. The cells were immunofluorescence stained. Pictures were taken with a 100× oil lens on the Nikon A1 Laser Scanning Confocal Microscope. The data show the effect of different EGFR ligands on the nuclear translocation of EGFRwt and EGFRmut. Pictures were taken with a 100× oil lens on the Nikon A1 laser scanning confocal microscope. The data are representative of three independent experiments.

    Techniques Used: Western Blot, Expressing, Concentration Assay, SDS Page, Activation Assay, Immunofluorescence, Staining, Microscopy, Translocation Assay

    24) Product Images from "Lnc RNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression, et al. LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression"

    Article Title: Lnc RNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression, et al. LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14085

    DUXAP 9‐206 interacts with Cbl‐b to reduce the degradation of EGFR . (A) The interaction between EGFR and Cbl‐b with DUXAP 9‐206 overexpression or knockdown was assessed by Co‐ IP assays. (B) Western blotting analysis of the expression levels of Cbl‐b and the EGFR signaling pathway downstream molecules p‐ AKT , AKT , p‐ ERK and ERK with ectopic expression or silencing of DUXAP 9‐206. Quantification of invading cells (C) and colony formation ability (D) in vector‐ and DUXAP 9‐206‐overexpressing cells by silencing of EGFR
    Figure Legend Snippet: DUXAP 9‐206 interacts with Cbl‐b to reduce the degradation of EGFR . (A) The interaction between EGFR and Cbl‐b with DUXAP 9‐206 overexpression or knockdown was assessed by Co‐ IP assays. (B) Western blotting analysis of the expression levels of Cbl‐b and the EGFR signaling pathway downstream molecules p‐ AKT , AKT , p‐ ERK and ERK with ectopic expression or silencing of DUXAP 9‐206. Quantification of invading cells (C) and colony formation ability (D) in vector‐ and DUXAP 9‐206‐overexpressing cells by silencing of EGFR

    Techniques Used: Over Expression, Co-Immunoprecipitation Assay, Western Blot, Expressing, Plasmid Preparation

    Clinical relevance of DUXAP 9‐206 with EGFR , p‐ EGFR , p‐ AKT and p‐ ERK in clinical specimens. (A) ISH analysis of DUXAP 9‐206 and immunohistochemical analysis of p‐ EGFR , EGFR , p‐ AKT and p‐ ERK expression in NSCLC tumour specimens. Sections were H E stained to visualize the tumour structure and boundaries. Original magnification, ×400. (B) DUXAP 9‐206 expression was positively associated with expression levels of p‐ EGFR , EGFR , p‐ AKT and p‐ ERK in NSCLC specimens
    Figure Legend Snippet: Clinical relevance of DUXAP 9‐206 with EGFR , p‐ EGFR , p‐ AKT and p‐ ERK in clinical specimens. (A) ISH analysis of DUXAP 9‐206 and immunohistochemical analysis of p‐ EGFR , EGFR , p‐ AKT and p‐ ERK expression in NSCLC tumour specimens. Sections were H E stained to visualize the tumour structure and boundaries. Original magnification, ×400. (B) DUXAP 9‐206 expression was positively associated with expression levels of p‐ EGFR , EGFR , p‐ AKT and p‐ ERK in NSCLC specimens

    Techniques Used: In Situ Hybridization, Immunohistochemistry, Expressing, Staining

    25) Product Images from "Cucurbitacin B and SCH772984 exhibit synergistic anti-pancreatic cancer activities by suppressing EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling"

    Article Title: Cucurbitacin B and SCH772984 exhibit synergistic anti-pancreatic cancer activities by suppressing EGFR, PI3K/Akt/mTOR, STAT3 and ERK signaling

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21704

    Proposed model of synergistic anti-pancreatic cancer activities of CuB and SCH772984 CuB suppresses EGFR levels, activity and downstream PI3K/Akt/mTOR and STAT3 signaling, but enhances ERK activity via AMPK activation, which inactivates the canonical apoptosis pathway. Addition of SCH772984 reverses ERK phosphorylation induced by CuB, thereby resulting in apoptosis.
    Figure Legend Snippet: Proposed model of synergistic anti-pancreatic cancer activities of CuB and SCH772984 CuB suppresses EGFR levels, activity and downstream PI3K/Akt/mTOR and STAT3 signaling, but enhances ERK activity via AMPK activation, which inactivates the canonical apoptosis pathway. Addition of SCH772984 reverses ERK phosphorylation induced by CuB, thereby resulting in apoptosis.

    Techniques Used: Activity Assay, Activation Assay

    CuB suppresses EGFR levels and downstream PI3K/Akt/mTOR and STAT3 signaling ( A ) BxPC-3 and HPAC cells were treated with vehicle control or CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with the indicated antibodies. ( B ) BxPC-3 and HPAC cells were treated with 0.3 µM CuB for up to 24 h. Cells were harvested and lysed. Protein extracts were analyzed by Western blotting and probed with the indicated antibodies. ( C and D ) BxPC-3 and HPAC cells were infected with EGFR (EGFR-shRNA) or non-target control shRNA lentivirus (NTC-shRNA). EGFR-shRNA or NTC-shRNA groups were cultured with CuB for 24 h. Cell viability was determined daily using the MTT assay. Data are presented as the mean ± standard error from at least 3 independent experiments. Statistical significance was calculated using the pair-wise 2-sample t -test. *** indicates p
    Figure Legend Snippet: CuB suppresses EGFR levels and downstream PI3K/Akt/mTOR and STAT3 signaling ( A ) BxPC-3 and HPAC cells were treated with vehicle control or CuB for 24 h. Whole cell lysates were analyzed by Western blotting and probed with the indicated antibodies. ( B ) BxPC-3 and HPAC cells were treated with 0.3 µM CuB for up to 24 h. Cells were harvested and lysed. Protein extracts were analyzed by Western blotting and probed with the indicated antibodies. ( C and D ) BxPC-3 and HPAC cells were infected with EGFR (EGFR-shRNA) or non-target control shRNA lentivirus (NTC-shRNA). EGFR-shRNA or NTC-shRNA groups were cultured with CuB for 24 h. Cell viability was determined daily using the MTT assay. Data are presented as the mean ± standard error from at least 3 independent experiments. Statistical significance was calculated using the pair-wise 2-sample t -test. *** indicates p

    Techniques Used: Western Blot, Infection, shRNA, Cell Culture, MTT Assay

    26) Product Images from "Rhythmic Pressure Waves Induce Mucin5AC Expression via an EGFR-Mediated Signaling Pathway in Human Airway Epithelial Cells"

    Article Title: Rhythmic Pressure Waves Induce Mucin5AC Expression via an EGFR-Mediated Signaling Pathway in Human Airway Epithelial Cells

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2013.2079

    The effect of RPW on the activation of EGFR, ERK1/2, and Akt in 16HBE cells. Cell lysates were prepared after 2 h of RPW exposure; p-EGFR (A) , p-ERK1/2 (B) , and p-Akt (C) protein expressions were measured by Western blotting analyses using specific
    Figure Legend Snippet: The effect of RPW on the activation of EGFR, ERK1/2, and Akt in 16HBE cells. Cell lysates were prepared after 2 h of RPW exposure; p-EGFR (A) , p-ERK1/2 (B) , and p-Akt (C) protein expressions were measured by Western blotting analyses using specific

    Techniques Used: Activation Assay, Western Blot

    The role of EGFR in RPW induced the phosphorylation of ERK1/2 and Akt. The cells were transfected with a siNT or specific siRNA for EGFR (siEGFR) before applying RPW. Cell lysates were prepared after 2 h of RPW exposure; p-ERK1/2 (A) and p-Akt
    Figure Legend Snippet: The role of EGFR in RPW induced the phosphorylation of ERK1/2 and Akt. The cells were transfected with a siNT or specific siRNA for EGFR (siEGFR) before applying RPW. Cell lysates were prepared after 2 h of RPW exposure; p-ERK1/2 (A) and p-Akt

    Techniques Used: Transfection

    27) Product Images from "The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M"

    Article Title: The role of c-Src in the invasion and metastasis of hepatocellular carcinoma cells induced by association of cell surface GRP78 with activated α2M

    Journal: BMC Cancer

    doi: 10.1186/s12885-015-1401-z

    Association of Cell surface GRP78 with α2M* facilitated the maximal activation of EGFR in a c-Src dependent manner. (a) Western blot analysis of the expression and phosphorylation of EGFR in serum starved QGY-7703 treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (b) Quantitative analysis of the expression and phosphorylation of EGFR in serum starved QGY-7703 treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (c) Co-immunoprecipitation analysis of the interaction between c-Src and EGFR in serum starved QGY-7703 cells treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (d) Quantitative analysis of the interaction between c-Src and EGFR in serum starved QGY-7703 cells treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*
    Figure Legend Snippet: Association of Cell surface GRP78 with α2M* facilitated the maximal activation of EGFR in a c-Src dependent manner. (a) Western blot analysis of the expression and phosphorylation of EGFR in serum starved QGY-7703 treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (b) Quantitative analysis of the expression and phosphorylation of EGFR in serum starved QGY-7703 treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (c) Co-immunoprecipitation analysis of the interaction between c-Src and EGFR in serum starved QGY-7703 cells treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*. (d) Quantitative analysis of the interaction between c-Src and EGFR in serum starved QGY-7703 cells treated with vehicle, α2M*, PP2 or PP2 in combination with α2M*

    Techniques Used: Activation Assay, Western Blot, Expressing, Immunoprecipitation

    28) Product Images from "Expression of LRIG1, a Negative Regulator of EGFR, Is Dynamically Altered during Different Stages of Gastric Carcinogenesis"

    Article Title: Expression of LRIG1, a Negative Regulator of EGFR, Is Dynamically Altered during Different Stages of Gastric Carcinogenesis

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2018.08.006

    Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on SNU638 xenografts and Kaplan-Meier survival curves of relapse-free survival (RFS) of gastric cancer patients with low or high LRIG1 mRNA expression. SNU638 cells, with or without vector transfection, were injected s.c. into the dorsal flank of 7-week-old male athymic nude mice to establish primary tumors. A: Tumor volumes were measured using calipers. B:  Extent of tumor growth was evaluated in vivo . Dotted circles represent tumor mass. C: Histopathologic analysis of gastric tumor masses from mouse xenograft model. Hematoxylin and eosin (H E) staining was performed to investigate epidermal growth factor receptor (EGFR) and phospho (p)-EGFR (Tyr1068) expression in tumor tissues from xenograft mice. Boxed areas are shown at higher magnification to the right. EGFR- and pEGFR-positive cells were counted from five microscopic fields. Statistical analyses were performed using JMP software version 4. D: Kaplan-Meier curves show RFS in gastric cancer patients, monitored for 150 months, based on high or low tumor LRIG1 mRNA expression levels. Red, patients with expression levels above the median; black, patients with expression levels below the median. Patients with low expression levels had a lower probability of RFS over time. Data are expressed as means ± SEM. n = 4 mice per treatment. ∗ P ÂÂ
    Figure Legend Snippet: Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on SNU638 xenografts and Kaplan-Meier survival curves of relapse-free survival (RFS) of gastric cancer patients with low or high LRIG1 mRNA expression. SNU638 cells, with or without vector transfection, were injected s.c. into the dorsal flank of 7-week-old male athymic nude mice to establish primary tumors. A: Tumor volumes were measured using calipers. B:  Extent of tumor growth was evaluated in vivo . Dotted circles represent tumor mass. C: Histopathologic analysis of gastric tumor masses from mouse xenograft model. Hematoxylin and eosin (H E) staining was performed to investigate epidermal growth factor receptor (EGFR) and phospho (p)-EGFR (Tyr1068) expression in tumor tissues from xenograft mice. Boxed areas are shown at higher magnification to the right. EGFR- and pEGFR-positive cells were counted from five microscopic fields. Statistical analyses were performed using JMP software version 4. D: Kaplan-Meier curves show RFS in gastric cancer patients, monitored for 150 months, based on high or low tumor LRIG1 mRNA expression levels. Red, patients with expression levels above the median; black, patients with expression levels below the median. Patients with low expression levels had a lower probability of RFS over time. Data are expressed as means ± SEM. n = 4 mice per treatment. ∗ P ÂÂ

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Injection, Mouse Assay, Staining, Software

    Immunohistochemistry (IHC) analysis of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 and epidermal growth factor receptor (EGFR) expression in human gastric preneoplastic and cancer tissues. LRIG1 and EGFR were detected in human gastric preneoplastic and cancer tissues using IHC with indicated antibodies. A and B: In a spasmolytic polypeptide–expressing metaplastic (SPEM) region ( A ), LRIG1 was expressed strongly, whereas EGFR expression was low along SPEM glands ( arrowheads ). C–E: Decreased LRIG1 expression ( C and D ; yellow arrowheads ) and low EGFR gland expression ( C  and D ; black arrowheads ) are observed in the intestinal metaplastic (IM) region, and LRIG1 is absent in cancer tissues ( E ). D and F: Strong EGFR expression in the IM region ( D , yellow arrowheads ) and in cancer lesions ( F ) is shown. Samples from five patients were used in these experiments G: LRIG1- or EGFR-positive cells were counted from 10 microscopic fields. Statistical analyses were performed using JMP software version 4. Data are expressed as means ± SEM. ∗ P ÂÂ
    Figure Legend Snippet: Immunohistochemistry (IHC) analysis of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 and epidermal growth factor receptor (EGFR) expression in human gastric preneoplastic and cancer tissues. LRIG1 and EGFR were detected in human gastric preneoplastic and cancer tissues using IHC with indicated antibodies. A and B: In a spasmolytic polypeptide–expressing metaplastic (SPEM) region ( A ), LRIG1 was expressed strongly, whereas EGFR expression was low along SPEM glands ( arrowheads ). C–E: Decreased LRIG1 expression ( C and D ; yellow arrowheads ) and low EGFR gland expression ( C  and D ; black arrowheads ) are observed in the intestinal metaplastic (IM) region, and LRIG1 is absent in cancer tissues ( E ). D and F: Strong EGFR expression in the IM region ( D , yellow arrowheads ) and in cancer lesions ( F ) is shown. Samples from five patients were used in these experiments G: LRIG1- or EGFR-positive cells were counted from 10 microscopic fields. Statistical analyses were performed using JMP software version 4. Data are expressed as means ± SEM. ∗ P ÂÂ

    Techniques Used: Immunohistochemistry, Expressing, Software

    Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on tumorigenesis in the SNU638 gastric cancer cell line. A: LRIG1 was depleted using pGIPZ-based shRNA (sh46 and sh71). Levels of LRIG1, epidermal growth factor receptor (EGFR), phospho-EGFR, and epithelial to mesenchymal markers [E-cadherin, occludin, N-cadherin, vimentin, zinc finger E box–binding homeobox (ZEB)-1/T-cell factor (TCF)-3, and anti–zinc finger protein SNAI1 (SNAIL)] were detected by Western blot analysis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. B: Cell proliferation was assessed with cell counting over 5 days. C: Cell cycle was examined with flow cytometry. D: Representative image of invasion and migration analyzed using Boyden chambers. E: Quantitative-analytic data from four independent experiments. Data are expressed as means ± SEM. n = 3 ( C ); n  = 4 ( A , B , and D ). ∗ P ÂÂ
    Figure Legend Snippet: Effects of leucine-rich repeats and immunoglobulin-like domains protein (LRIG)-1 knockdown on tumorigenesis in the SNU638 gastric cancer cell line. A: LRIG1 was depleted using pGIPZ-based shRNA (sh46 and sh71). Levels of LRIG1, epidermal growth factor receptor (EGFR), phospho-EGFR, and epithelial to mesenchymal markers [E-cadherin, occludin, N-cadherin, vimentin, zinc finger E box–binding homeobox (ZEB)-1/T-cell factor (TCF)-3, and anti–zinc finger protein SNAI1 (SNAIL)] were detected by Western blot analysis using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. B: Cell proliferation was assessed with cell counting over 5 days. C: Cell cycle was examined with flow cytometry. D: Representative image of invasion and migration analyzed using Boyden chambers. E: Quantitative-analytic data from four independent experiments. Data are expressed as means ± SEM. n = 3 ( C ); n  = 4 ( A , B , and D ). ∗ P ÂÂ

    Techniques Used: shRNA, Western Blot, Cell Counting, Flow Cytometry, Cytometry, Migration

    29) Product Images from "Cells with intense EGFR staining and a high nuclear to cytoplasmic ratio are specific for infiltrative glioma: a useful marker in neuropathological practice"

    Article Title: Cells with intense EGFR staining and a high nuclear to cytoplasmic ratio are specific for infiltrative glioma: a useful marker in neuropathological practice

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/not094

    (A) Characterization of strongly EGFR-positive cells in infiltrative glioma tissue sections. Double-staining was performed on paraffin-embedded tissue section of infiltrative glioma using anti-EGFR antibody (green), and (a) anti-GFAP antibody (red) or (b) anti-olig2 antibody (red), or (c) anti-nestin antibody (red) (×400). Strongly EGFR-stained cells with a high nucleus to cytoplasm ratio lacked GFAP expression (a) and coexpressed olig2 (b) and nestin (c). (B and C) Characterization of EGFR-overexpressing cells sorted from fresh glioblastoma. Immunofluorescence performed immediately after FACS sorting of small EGFR-overexpressing cells (B) confirmed that cells overexpressed EGFR and showed that these cells expressed the neural stem cell markers oct4, sox1, sox2, and A2B5 and (C) expressed invasion marker CXCR4 and CD44 (×1000). (D) Culture of small EGFR overexpressing cells in a medium specific for neural stem cells (×1000). Cells slowly proliferated and formed small neurospheres (left) or remained nonadhesive quiescent isolated cells (right).
    Figure Legend Snippet: (A) Characterization of strongly EGFR-positive cells in infiltrative glioma tissue sections. Double-staining was performed on paraffin-embedded tissue section of infiltrative glioma using anti-EGFR antibody (green), and (a) anti-GFAP antibody (red) or (b) anti-olig2 antibody (red), or (c) anti-nestin antibody (red) (×400). Strongly EGFR-stained cells with a high nucleus to cytoplasm ratio lacked GFAP expression (a) and coexpressed olig2 (b) and nestin (c). (B and C) Characterization of EGFR-overexpressing cells sorted from fresh glioblastoma. Immunofluorescence performed immediately after FACS sorting of small EGFR-overexpressing cells (B) confirmed that cells overexpressed EGFR and showed that these cells expressed the neural stem cell markers oct4, sox1, sox2, and A2B5 and (C) expressed invasion marker CXCR4 and CD44 (×1000). (D) Culture of small EGFR overexpressing cells in a medium specific for neural stem cells (×1000). Cells slowly proliferated and formed small neurospheres (left) or remained nonadhesive quiescent isolated cells (right).

    Techniques Used: Double Staining, Staining, Expressing, Immunofluorescence, FACS, Marker, Isolation

    30) Product Images from "Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines"

    Article Title: Exogenous HGF Bypasses the Effects of ErbB Inhibition on Tumor Cell Viability in Medulloblastoma Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0141381

    HGF and EGF phosphorylate MET and EGFR, respectively. A-B Immunoblots, showing the effects of A HGF stimulation (0-100ng/ml) in combination with crizotinib on MET (MET: 145kDa, pro-MET: 170kDa) phosphorylation and B EGF stimulation (0-100ng/ml) in combination with canertinib on EGFR (175kDa) phosphorylation in cell lines RES256 and UW473. Bands of MET represent MET (145kDa) and pro-MET (170kDa). Total-MET and total-EGFR were used as loading controls. C-D Quantification analysis of immunoblots showing the C normalized phospho-MET expression and D normalized phospho-EGFR expression. Quantitations are representative of 2 to 3 independent experiments. Student’s t-test: *p
    Figure Legend Snippet: HGF and EGF phosphorylate MET and EGFR, respectively. A-B Immunoblots, showing the effects of A HGF stimulation (0-100ng/ml) in combination with crizotinib on MET (MET: 145kDa, pro-MET: 170kDa) phosphorylation and B EGF stimulation (0-100ng/ml) in combination with canertinib on EGFR (175kDa) phosphorylation in cell lines RES256 and UW473. Bands of MET represent MET (145kDa) and pro-MET (170kDa). Total-MET and total-EGFR were used as loading controls. C-D Quantification analysis of immunoblots showing the C normalized phospho-MET expression and D normalized phospho-EGFR expression. Quantitations are representative of 2 to 3 independent experiments. Student’s t-test: *p

    Techniques Used: Western Blot, Expressing

    31) Product Images from "SNX15 links clathrin endocytosis to the PtdIns3P early endosome independently of the APPL1 endosome"

    Article Title: SNX15 links clathrin endocytosis to the PtdIns3P early endosome independently of the APPL1 endosome

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.125732

    SNX15 decorated puncta colocalise with clathrin. HeLa cells expressing GFP–SNX15 were stimulated with 100 ng/ml EGF for the indicated times. Fixed cells were co-immunostained using antibodies against clathrin and EGFR, and imaged using
    Figure Legend Snippet: SNX15 decorated puncta colocalise with clathrin. HeLa cells expressing GFP–SNX15 were stimulated with 100 ng/ml EGF for the indicated times. Fixed cells were co-immunostained using antibodies against clathrin and EGFR, and imaged using

    Techniques Used: Expressing

    SNX15 regulates trafficking of internalised EGFR. HeLa cells transfected with scrambled or SNX15 siRNA were stimulated with 100 ng/ml EGF (A,B,D,E) or Alexa-Fluor-488–EGF (C) for the indicated time periods. (A) Flow cytometric analysis
    Figure Legend Snippet: SNX15 regulates trafficking of internalised EGFR. HeLa cells transfected with scrambled or SNX15 siRNA were stimulated with 100 ng/ml EGF (A,B,D,E) or Alexa-Fluor-488–EGF (C) for the indicated time periods. (A) Flow cytometric analysis

    Techniques Used: Transfection, Flow Cytometry

    32) Product Images from "Linc00152 suppresses apoptosis and promotes migration by sponging miR-4767 in vascular endothelial cells"

    Article Title: Linc00152 suppresses apoptosis and promotes migration by sponging miR-4767 in vascular endothelial cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18777

    MiR-4767 directly targets Bcl2L12 and EGFR (A) Potential sited targeted by miR-4767 in the sequences of Bcl2L12 and EGFR 3’UTRs. (B) wt and mut sequences of Bcl2L12 or EGFR inserted into the luciferase reporter gene vectors. Luc-Bcl2L12 3’UTR-wt or Luc-Bcl2L12 3’UTR-mut vectors (C) and Luc-EGFR 3’UTR-wt or Luc-EGFR 3’UTR-mut vectors (D) were co-transfected into HEK293T cells with 40 nM miR-4767 mimics or NC mimics for 48 h and luciferase activity was measured. ** P
    Figure Legend Snippet: MiR-4767 directly targets Bcl2L12 and EGFR (A) Potential sited targeted by miR-4767 in the sequences of Bcl2L12 and EGFR 3’UTRs. (B) wt and mut sequences of Bcl2L12 or EGFR inserted into the luciferase reporter gene vectors. Luc-Bcl2L12 3’UTR-wt or Luc-Bcl2L12 3’UTR-mut vectors (C) and Luc-EGFR 3’UTR-wt or Luc-EGFR 3’UTR-mut vectors (D) were co-transfected into HEK293T cells with 40 nM miR-4767 mimics or NC mimics for 48 h and luciferase activity was measured. ** P

    Techniques Used: Luciferase, Transfection, Activity Assay

    Linc00152 promoted the expression of Bcl2L12 and EGFR through diminishing miR-4767 (A) MiR-4767 negatively regulated the protein levels of Bcl2L12 and EGFR. HUVECs were transfected with miR-4767 mimics at concentrations of 20, and 40 nM or miR-4767 antagomirs at 10 and 20 nM for 48 h. Then, the cells were treated with 150 μg/mL ox-LDL for 24 h and the protein levels of Bcl2L12 and EGFR were detected by Western blotting. (B) Linc00152 promoted the expression of Bcl2L12 and EGFR through diminishing miR-4767. (C and D) Block of miR-4767 antagonized the changes of apoptosis and migration in HUVECs caused by linc00152 knockdown. HUVECs were transfected with 1.0 μg/mL pcDNA3.1-linc00152, 40 nM Linc00152 siRNA, or 40 nM Linc00152-siRNA plus 20 nM miR-4767 antagomirs. After incubation for 48 h, the cells were treated with 150 μg/mL ox-LDL for 24 h. The protein levels of Bcl2L12 and EGFR were detected by Western blotting. Cell apoptosis was checked by TUNEL assay and cell migration was detected with the Transwell Migration assay. * P
    Figure Legend Snippet: Linc00152 promoted the expression of Bcl2L12 and EGFR through diminishing miR-4767 (A) MiR-4767 negatively regulated the protein levels of Bcl2L12 and EGFR. HUVECs were transfected with miR-4767 mimics at concentrations of 20, and 40 nM or miR-4767 antagomirs at 10 and 20 nM for 48 h. Then, the cells were treated with 150 μg/mL ox-LDL for 24 h and the protein levels of Bcl2L12 and EGFR were detected by Western blotting. (B) Linc00152 promoted the expression of Bcl2L12 and EGFR through diminishing miR-4767. (C and D) Block of miR-4767 antagonized the changes of apoptosis and migration in HUVECs caused by linc00152 knockdown. HUVECs were transfected with 1.0 μg/mL pcDNA3.1-linc00152, 40 nM Linc00152 siRNA, or 40 nM Linc00152-siRNA plus 20 nM miR-4767 antagomirs. After incubation for 48 h, the cells were treated with 150 μg/mL ox-LDL for 24 h. The protein levels of Bcl2L12 and EGFR were detected by Western blotting. Cell apoptosis was checked by TUNEL assay and cell migration was detected with the Transwell Migration assay. * P

    Techniques Used: Expressing, Transfection, Western Blot, Blocking Assay, Migration, Incubation, TUNEL Assay, Transwell Migration Assay

    33) Product Images from "Phosphoproteomic analysis reveals Smarcb1 dependent EGFR signaling in Malignant Rhabdoid tumor cells"

    Article Title: Phosphoproteomic analysis reveals Smarcb1 dependent EGFR signaling in Malignant Rhabdoid tumor cells

    Journal: Molecular Cancer

    doi: 10.1186/s12943-015-0439-5

    EGFR activation mediates AKT activation in Smarcb1 deficient cells. a Inhibition of AKT activation upon treatment with the EGFR/HER2 inhibitor Lapatinib (Lap.) and the EGFR inhibitor Gefitinib (Gef.) versus the DMSO (D.) control. b WST1 proliferation assay demonstrating relative proliferation of Smarcb1 deficient and proficient cells following a 6 day treatment with the EGFR inhibitors Lapatinib/Gefitinib, with the AKT inhibitor 1/2, dual inhibition and serum withdrawal
    Figure Legend Snippet: EGFR activation mediates AKT activation in Smarcb1 deficient cells. a Inhibition of AKT activation upon treatment with the EGFR/HER2 inhibitor Lapatinib (Lap.) and the EGFR inhibitor Gefitinib (Gef.) versus the DMSO (D.) control. b WST1 proliferation assay demonstrating relative proliferation of Smarcb1 deficient and proficient cells following a 6 day treatment with the EGFR inhibitors Lapatinib/Gefitinib, with the AKT inhibitor 1/2, dual inhibition and serum withdrawal

    Techniques Used: Activation Assay, Inhibition, Proliferation Assay

    34) Product Images from "miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6"

    Article Title: miR-2861 acts as a tumor suppressor via targeting EGFR/AKT2/CCND1 pathway in cervical cancer induced by human papillomavirus virus 16 E6

    Journal: Scientific Reports

    doi: 10.1038/srep28968

    Knockdown of EGFR, AKT2, or CCND1 produces similar suppressive effects to that of miR-2861 overexpression in cervical cancer cells. ( A ) Knockdown of EGFR, AKT2, or CCND1 suppresses cell proliferation of both SiHa and CaSki cells. CCK8 assay was performed to determine the growth of SiHa and CaSki cells treated with si-EGFR, si-AKT2, si-CCND1, or si-NS. ( B , C ) Knockdown of EGFR, AKT2, or CCND1 enhances cell apoptosis of SiHa and CaSki cells. Apoptosis assay was determined in SiHa and CaSki cells at 48 h after transfection of si-EGFR, si-AKT2, si-CCND1, or si-NS. Representative images are shown ( C ) and early apoptosis rate are indicated ( B ). ** P
    Figure Legend Snippet: Knockdown of EGFR, AKT2, or CCND1 produces similar suppressive effects to that of miR-2861 overexpression in cervical cancer cells. ( A ) Knockdown of EGFR, AKT2, or CCND1 suppresses cell proliferation of both SiHa and CaSki cells. CCK8 assay was performed to determine the growth of SiHa and CaSki cells treated with si-EGFR, si-AKT2, si-CCND1, or si-NS. ( B , C ) Knockdown of EGFR, AKT2, or CCND1 enhances cell apoptosis of SiHa and CaSki cells. Apoptosis assay was determined in SiHa and CaSki cells at 48 h after transfection of si-EGFR, si-AKT2, si-CCND1, or si-NS. Representative images are shown ( C ) and early apoptosis rate are indicated ( B ). ** P

    Techniques Used: Over Expression, CCK-8 Assay, Apoptosis Assay, Transfection

    miR-2861 targets EGFR, AKT2, and CCND1 in cervical cancer cells. ( A ) Predicted EGFR, AKT2, and CCND1 3′ UTR binding sites for miR-2861. The alignment of the seed region of miR-2861 with EGFR, AKT2, and CCND1 3′UTR are shown. The mutated sites of targets are indicated in red. ( B ) EGFR 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) EGFR 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells and the luciferase reporter assay was performed at 24 h posttransfection. ( C ) AKT2 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing each wt (wt1, 2, 3, 4, and 5) or mutated (mut1, 2, 3, 4, and 5) AKT2 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells, respectively. The luciferase assay was then performed. ( D ) CCND1 3′UTR is also a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) CCND1 3′UTR was cotransfected with miR-2861 or miR-NC and the luciferase assay was performed. ( E ) miR-2861 overexpression decreased endogenous levels of EGFR, AKT2, and CCND1 proteins in SiHa and CaSki cells. SiHa and CaSki cells were transfected with miR-2861 or miR-NC for 72 h, respectively. EGFR, AKT2, and CCND1 expressions were assessed by Western blot. GAPDH was obtained as a loading control. Error bars, ± SD. * P
    Figure Legend Snippet: miR-2861 targets EGFR, AKT2, and CCND1 in cervical cancer cells. ( A ) Predicted EGFR, AKT2, and CCND1 3′ UTR binding sites for miR-2861. The alignment of the seed region of miR-2861 with EGFR, AKT2, and CCND1 3′UTR are shown. The mutated sites of targets are indicated in red. ( B ) EGFR 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) EGFR 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells and the luciferase reporter assay was performed at 24 h posttransfection. ( C ) AKT2 3′UTR is a target of miR-2861. pmiR-GLO luciferase construct containing each wt (wt1, 2, 3, 4, and 5) or mutated (mut1, 2, 3, 4, and 5) AKT2 3′UTR was cotransfected with miR-2861 or miR-NC in SiHa cells, respectively. The luciferase assay was then performed. ( D ) CCND1 3′UTR is also a target of miR-2861. pmiR-GLO luciferase construct containing a wt (left) or mutated (right) CCND1 3′UTR was cotransfected with miR-2861 or miR-NC and the luciferase assay was performed. ( E ) miR-2861 overexpression decreased endogenous levels of EGFR, AKT2, and CCND1 proteins in SiHa and CaSki cells. SiHa and CaSki cells were transfected with miR-2861 or miR-NC for 72 h, respectively. EGFR, AKT2, and CCND1 expressions were assessed by Western blot. GAPDH was obtained as a loading control. Error bars, ± SD. * P

    Techniques Used: Binding Assay, Luciferase, Construct, Reporter Assay, Over Expression, Transfection, Western Blot

    Knockdown of EGFR, AKT2, or CCND1 suppresses cell invasion and increased E-cadherin level in cervical cancer cells. ( A , B ) A matrigel invasion assay was performed in SiHa ( A ) and CaSki ( B ) cells transfected with si-EGFR, si-AKT2, si-CCND1, or si-NS. Representative images are shown (magnification: ×200). Error bars indicate ± SD. ( C ) Suppression of EGFR, AKT2, or CCND1 increased E-cadherin expression level in both SiHa and CaSki cells. 72 h after transfection, the protein level of E-cadherin was analyzed by Western blot. ** P
    Figure Legend Snippet: Knockdown of EGFR, AKT2, or CCND1 suppresses cell invasion and increased E-cadherin level in cervical cancer cells. ( A , B ) A matrigel invasion assay was performed in SiHa ( A ) and CaSki ( B ) cells transfected with si-EGFR, si-AKT2, si-CCND1, or si-NS. Representative images are shown (magnification: ×200). Error bars indicate ± SD. ( C ) Suppression of EGFR, AKT2, or CCND1 increased E-cadherin expression level in both SiHa and CaSki cells. 72 h after transfection, the protein level of E-cadherin was analyzed by Western blot. ** P

    Techniques Used: Invasion Assay, Transfection, Expressing, Western Blot

    EGFR, AKT2, and CCND1 rescue miR-2861-induced cellular phenotypes in SiHa cells. Cells were cotransfected with EGFR, AKT2, CCND1 or empty vector and miR-2861 or miR-NC. ( A ) Overexpression of either EGFR, AKT2, or CCND1 recovers miR-2861-induced cell proliferation. CCK8 assay was assessed at 72 h after cotransfection. ( B ) Overexpression of either EGFR, AKT2, or CCND1 rescues miR-2861-induced inhibition of cell invasion. Representative images are shown (magnification: ×200). Error bars indicate ± SD. * P
    Figure Legend Snippet: EGFR, AKT2, and CCND1 rescue miR-2861-induced cellular phenotypes in SiHa cells. Cells were cotransfected with EGFR, AKT2, CCND1 or empty vector and miR-2861 or miR-NC. ( A ) Overexpression of either EGFR, AKT2, or CCND1 recovers miR-2861-induced cell proliferation. CCK8 assay was assessed at 72 h after cotransfection. ( B ) Overexpression of either EGFR, AKT2, or CCND1 rescues miR-2861-induced inhibition of cell invasion. Representative images are shown (magnification: ×200). Error bars indicate ± SD. * P

    Techniques Used: Plasmid Preparation, Over Expression, CCK-8 Assay, Cotransfection, Inhibition

    35) Product Images from "Lnc RNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression, et al. LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression"

    Article Title: Lnc RNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression, et al. LncRNA DUXAP9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.14085

    Proposed functional action of DUXAP 9‐206 in modulating NSCLC proliferation and metastasis. Lnc RNA DUXAP 9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression
    Figure Legend Snippet: Proposed functional action of DUXAP 9‐206 in modulating NSCLC proliferation and metastasis. Lnc RNA DUXAP 9‐206 directly binds with Cbl‐b to augment EGFR signaling and promotes non‐small cell lung cancer progression

    Techniques Used: Functional Assay

    DUXAP 9‐206 interacts with Cbl‐b to reduce the degradation of EGFR . (A) The interaction between EGFR and Cbl‐b with DUXAP 9‐206 overexpression or knockdown was assessed by Co‐ IP assays. (B) Western blotting analysis of the expression levels of Cbl‐b and the EGFR signaling pathway downstream molecules p‐ AKT , AKT , p‐ ERK and ERK with ectopic expression or silencing of DUXAP 9‐206. Quantification of invading cells (C) and colony formation ability (D) in vector‐ and DUXAP 9‐206‐overexpressing cells by silencing of EGFR
    Figure Legend Snippet: DUXAP 9‐206 interacts with Cbl‐b to reduce the degradation of EGFR . (A) The interaction between EGFR and Cbl‐b with DUXAP 9‐206 overexpression or knockdown was assessed by Co‐ IP assays. (B) Western blotting analysis of the expression levels of Cbl‐b and the EGFR signaling pathway downstream molecules p‐ AKT , AKT , p‐ ERK and ERK with ectopic expression or silencing of DUXAP 9‐206. Quantification of invading cells (C) and colony formation ability (D) in vector‐ and DUXAP 9‐206‐overexpressing cells by silencing of EGFR

    Techniques Used: Over Expression, Co-Immunoprecipitation Assay, Western Blot, Expressing, Plasmid Preparation

    36) Product Images from "EGFR Induces E2F1-Mediated Corticotroph Tumorigenesis"

    Article Title: EGFR Induces E2F1-Mediated Corticotroph Tumorigenesis

    Journal: Journal of the Endocrine Society

    doi: 10.1210/js.2016-1053

    Effect of gefitinib treatment in corti-EGFR-Tg mice and detection of E2F1. (a–b) Plasma ACTH and corticosterone levels in corti-EGFR-Tg mice before (left) and after (right) gefitinib or vehicle treatment. (c) Micro-MRI of corti-EGFR-Tg pituitary of pre- (left) and post- (right) gefitinib treatment. (d) Comparison of pituitary sizes pre- and post- gefitinib or vehicle treatment. (e) Pomc, EGFR, p -EGFR, p -Akt, total (t)-Akt, p -Erk, and t -Erk expression analyzed by immunoblotting using pituitary tumors derived from corti-EGFR-Tg treated with vehicle (left) or gefitinib (right). Quantifications are shown in the graph. (f) Glucose levels during IPGTT at 0, 30, 60, and 120 min in corti-EGFR-Tg pre- and posttreatment with vehicle (n = 5) or gefitinib (n = 5). (g) Pomc, EGFR, total E2F1, and pS337-E2F1 expression analyzed by immunoblotting using pituitary tissues derived from WT (left) and pituitary tumors derived from corti-EGFR-Tg (right). Quantifications are shown in the graph. (h) Pomc, EGFR, total E2F1, and pS337-E2F1 expression analyzed by immunoblotting in corti-EGFR-Tg pituitary tumors after vehicle (left) or gefitinib treatment (right). Quantifications are shown in the graph. * P
    Figure Legend Snippet: Effect of gefitinib treatment in corti-EGFR-Tg mice and detection of E2F1. (a–b) Plasma ACTH and corticosterone levels in corti-EGFR-Tg mice before (left) and after (right) gefitinib or vehicle treatment. (c) Micro-MRI of corti-EGFR-Tg pituitary of pre- (left) and post- (right) gefitinib treatment. (d) Comparison of pituitary sizes pre- and post- gefitinib or vehicle treatment. (e) Pomc, EGFR, p -EGFR, p -Akt, total (t)-Akt, p -Erk, and t -Erk expression analyzed by immunoblotting using pituitary tumors derived from corti-EGFR-Tg treated with vehicle (left) or gefitinib (right). Quantifications are shown in the graph. (f) Glucose levels during IPGTT at 0, 30, 60, and 120 min in corti-EGFR-Tg pre- and posttreatment with vehicle (n = 5) or gefitinib (n = 5). (g) Pomc, EGFR, total E2F1, and pS337-E2F1 expression analyzed by immunoblotting using pituitary tissues derived from WT (left) and pituitary tumors derived from corti-EGFR-Tg (right). Quantifications are shown in the graph. (h) Pomc, EGFR, total E2F1, and pS337-E2F1 expression analyzed by immunoblotting in corti-EGFR-Tg pituitary tumors after vehicle (left) or gefitinib treatment (right). Quantifications are shown in the graph. * P

    Techniques Used: Mouse Assay, Micro-MRI, Expressing, Derivative Assay

    37) Product Images from "Metalloproteinase dependent reduction of cell surface cluster determinants upon the induction of apoptosis"

    Article Title: Metalloproteinase dependent reduction of cell surface cluster determinants upon the induction of apoptosis

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2344

    Measurement of steady state mRNA expression for ACTB, HLA-A, EGFR and IGF1R. Data were accumulated by real-time RT-PCR. cDNA(s) were generated from RNA isolated from both MK886 (10 h) and vehicle control (DMSO) LN18 calls. PCR amplicons were generated with primer probes that were specific for ACTB, HLA-A, EGFR and IGF1R. The abscissae show the progression of cycles up to a maximum of 40. The intersections of the horizontal threshold lines indicate the number of cycles to threshold (Ct). The ordinate values indicate the fluorescence normalized to the value at 40 cycles.
    Figure Legend Snippet: Measurement of steady state mRNA expression for ACTB, HLA-A, EGFR and IGF1R. Data were accumulated by real-time RT-PCR. cDNA(s) were generated from RNA isolated from both MK886 (10 h) and vehicle control (DMSO) LN18 calls. PCR amplicons were generated with primer probes that were specific for ACTB, HLA-A, EGFR and IGF1R. The abscissae show the progression of cycles up to a maximum of 40. The intersections of the horizontal threshold lines indicate the number of cycles to threshold (Ct). The ordinate values indicate the fluorescence normalized to the value at 40 cycles.

    Techniques Used: Expressing, Quantitative RT-PCR, Generated, Isolation, Polymerase Chain Reaction, Fluorescence

    Apoptotic decreases in HLA, EGFR and IGF1R cell surface determinants. Composites (curves A, pink; curves B, purple; and curves C, green) of flow cytometry histograms showing phycoerythrin fluorescence intensity vs. cell count for the LN18 histocompatibility antigens HLA-ABC, and cell surface growth factor receptors EGFR and IGF1R. All curves were obtained by using mouse primary antibodies followed by goat anti-mouse-IgG conjugated to phycoerythrin. Curves labeled A (pink) show the non-specific labeling that was obtained by using mouse anti-KLH as a primary antibody. Curves labeled B (purple) indicate the fluorescence obtained by the mouse antibodies specific for the determinants noted in the abscissae for the non-treated vehicle control (DMSO) LN18 cells. Curves labeled C (green) indicate the fluorescence obtained by the mouse antibodies specific for each of the determinants noted in the abscissae for those LN18 cells treated with MK886 for 14 h.
    Figure Legend Snippet: Apoptotic decreases in HLA, EGFR and IGF1R cell surface determinants. Composites (curves A, pink; curves B, purple; and curves C, green) of flow cytometry histograms showing phycoerythrin fluorescence intensity vs. cell count for the LN18 histocompatibility antigens HLA-ABC, and cell surface growth factor receptors EGFR and IGF1R. All curves were obtained by using mouse primary antibodies followed by goat anti-mouse-IgG conjugated to phycoerythrin. Curves labeled A (pink) show the non-specific labeling that was obtained by using mouse anti-KLH as a primary antibody. Curves labeled B (purple) indicate the fluorescence obtained by the mouse antibodies specific for the determinants noted in the abscissae for the non-treated vehicle control (DMSO) LN18 cells. Curves labeled C (green) indicate the fluorescence obtained by the mouse antibodies specific for each of the determinants noted in the abscissae for those LN18 cells treated with MK886 for 14 h.

    Techniques Used: Flow Cytometry, Cytometry, Fluorescence, Cell Counting, Labeling

    38) Product Images from "s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide"

    Article Title: s-HBEGF/SIRT1 circuit-dictated crosstalk between vascular endothelial cells and keratinocytes mediates sorafenib-induced hand–foot skin reaction that can be reversed by nicotinamide

    Journal: Cell Research

    doi: 10.1038/s41422-020-0309-6

    Schematic representation of the mechanism underlying sorafenib-induced hyper-keratosis. Vascular endothelial cells release s-HBEGF upon sorafenib stimulation. s-HBEGF binds to EGFR and leads to JNK2 phosphorylation in keratinocytes. The activated JNK2 subsequently stabilizes SIRT1, which eventually results in keratinization. The classic SIRT1 inhibitor nicotinamide could effectively reverse sorafenib-induced HFSR.
    Figure Legend Snippet: Schematic representation of the mechanism underlying sorafenib-induced hyper-keratosis. Vascular endothelial cells release s-HBEGF upon sorafenib stimulation. s-HBEGF binds to EGFR and leads to JNK2 phosphorylation in keratinocytes. The activated JNK2 subsequently stabilizes SIRT1, which eventually results in keratinization. The classic SIRT1 inhibitor nicotinamide could effectively reverse sorafenib-induced HFSR.

    Techniques Used:

    The EGFR-JNK2 axis is involved in sorafenib-induced hyper-keratosis. a HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR . The transcription level of EGFR was detected by RT-qPCR (upper panel, n = 3) and the expression level of EGFR was determined by western blot (lower panel). b – e HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR , followed by treatment with CdM CTRL or CdM SORA for 24 h ( b , d ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( c , e ). The cell survival rates were measured by SRB assay ( n = 3) ( b , c ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( d , e ). f Relevant EGFR downstream signaling pathways were examined by western blot. g Human primary keratinocytes were treated with s-HBEGF and the expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. h HaCaT cells or human primary keratinocytes were treated with or without sorafenib, CdM CTRL or CdM SORA . The expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. i HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 . JNK2 transcription level was detected by RT-qPCR (upper panel, n = 3) and the expression level of JNK2 was determined by western blot (lower panel). j–m HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 , followed by treatment with CdM CTRL or CdM SORA for 24 h ( j , l ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( k , m ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( j , k ). The cell survival rates were measured by SRB assay ( n = 3) ( l , m ). Densitometric values are shown as optical density after ACTB or GAPDH normalization using Image J. The results in ( a ), ( b ), ( c ), ( d ), ( e ), ( i ), ( j ), ( k ), ( l ) and ( m ) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with Dunn’s post hoc test in ( a ), ( i ) and when comparing the levels of KRT1 in ( j ) and with LSD post hoc test in ( d ), ( e ), ( k ) and when comparing the levels of KRT10 , LORICRIN and IVL in ( j ). n.s. no significance; * P
    Figure Legend Snippet: The EGFR-JNK2 axis is involved in sorafenib-induced hyper-keratosis. a HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR . The transcription level of EGFR was detected by RT-qPCR (upper panel, n = 3) and the expression level of EGFR was determined by western blot (lower panel). b – e HaCaT cells were transfected with non-targeting siRNA or siRNA targeting EGFR , followed by treatment with CdM CTRL or CdM SORA for 24 h ( b , d ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( c , e ). The cell survival rates were measured by SRB assay ( n = 3) ( b , c ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( d , e ). f Relevant EGFR downstream signaling pathways were examined by western blot. g Human primary keratinocytes were treated with s-HBEGF and the expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. h HaCaT cells or human primary keratinocytes were treated with or without sorafenib, CdM CTRL or CdM SORA . The expression levels of p-JNK1/2, JNK2, p-JNK1 and JNK1 were assessed by western blot. i HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 . JNK2 transcription level was detected by RT-qPCR (upper panel, n = 3) and the expression level of JNK2 was determined by western blot (lower panel). j–m HaCaT cells were transfected with non-targeting siRNA or siRNA targeting JNK2 , followed by treatment with CdM CTRL or CdM SORA for 24 h ( j , l ) or treatment with or without s-HBEGF (2.5 ng/mL) for 24 h ( k , m ). The transcription levels of KRT1 , KRT10 , LORICRIN and IVL were measured by RT-qPCR ( n = 3) ( j , k ). The cell survival rates were measured by SRB assay ( n = 3) ( l , m ). Densitometric values are shown as optical density after ACTB or GAPDH normalization using Image J. The results in ( a ), ( b ), ( c ), ( d ), ( e ), ( i ), ( j ), ( k ), ( l ) and ( m ) are presented as the mean ± SD. Statistical analyses were performed using one-way ANOVA with Dunn’s post hoc test in ( a ), ( i ) and when comparing the levels of KRT1 in ( j ) and with LSD post hoc test in ( d ), ( e ), ( k ) and when comparing the levels of KRT10 , LORICRIN and IVL in ( j ). n.s. no significance; * P

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Sulforhodamine B Assay

    39) Product Images from "Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer"

    Article Title: Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E16-06-0427

    Combined MOR and SSTR2 agonist treatment leads to a distinct signaling pathway in PANC-1 cells. (A) After treatment of either normal epithelial pancreatic cells or PANC-1 cells with agonists, phosphorylation of ERK1/2 and EGFR in cell lysates was analyzed (Western blot detection). The agonist treatments were 10 nM dermorphin, 10 nM L-054,264, or 10 nM dermorphin with 10 nM L-054,264. Treatment time periods are indicated. Images were cropped for clarity; large regions of representative original images are provided in Supplemental Figure S13E. (B) Image Lab software was used to quantify the amount of ERK1/2 or EGFR phosphorylation in each lane. The data are expressed as a ratio of either pERK1/2 over total ERK1/2 or pEGFR over total EGFR and averaged. Results are normalized (the maximum response for dermorphin in PANC-1 cells is taken as 100%) and presented with SE. Dermorphin treatment is presented in purple; L-054,264 treatment is presented in red; and the combined treatment is presented in blue. * p
    Figure Legend Snippet: Combined MOR and SSTR2 agonist treatment leads to a distinct signaling pathway in PANC-1 cells. (A) After treatment of either normal epithelial pancreatic cells or PANC-1 cells with agonists, phosphorylation of ERK1/2 and EGFR in cell lysates was analyzed (Western blot detection). The agonist treatments were 10 nM dermorphin, 10 nM L-054,264, or 10 nM dermorphin with 10 nM L-054,264. Treatment time periods are indicated. Images were cropped for clarity; large regions of representative original images are provided in Supplemental Figure S13E. (B) Image Lab software was used to quantify the amount of ERK1/2 or EGFR phosphorylation in each lane. The data are expressed as a ratio of either pERK1/2 over total ERK1/2 or pEGFR over total EGFR and averaged. Results are normalized (the maximum response for dermorphin in PANC-1 cells is taken as 100%) and presented with SE. Dermorphin treatment is presented in purple; L-054,264 treatment is presented in red; and the combined treatment is presented in blue. * p

    Techniques Used: Western Blot, Software

    40) Product Images from "Using a Novel MicroRNA Delivery System to Inhibit Osteoclastogenesis"

    Article Title: Using a Novel MicroRNA Delivery System to Inhibit Osteoclastogenesis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms16048337

    Western blot analysis of EGFR and TRAF6 expression. Cells were stimulated with RANKL/M-CSF for three days and treated with 500 nM MS2-miR-146a VLPs (test group) or 500 nM MS2-miRNC VLPs (negative control group (NC)). Lysates were cleared of cellular debris, and equal concentrations of protein (30 µg) were separated via SDS-PAGE. Proteins were identified by incubating polyvinylidene fluoride (PVDF) membrane with monoclonal antibodies. β-actin was used as a loading control. ( A , B ) Expression level of EGFR; and ( C , D ) Expression level of TRAF6. * p
    Figure Legend Snippet: Western blot analysis of EGFR and TRAF6 expression. Cells were stimulated with RANKL/M-CSF for three days and treated with 500 nM MS2-miR-146a VLPs (test group) or 500 nM MS2-miRNC VLPs (negative control group (NC)). Lysates were cleared of cellular debris, and equal concentrations of protein (30 µg) were separated via SDS-PAGE. Proteins were identified by incubating polyvinylidene fluoride (PVDF) membrane with monoclonal antibodies. β-actin was used as a loading control. ( A , B ) Expression level of EGFR; and ( C , D ) Expression level of TRAF6. * p

    Techniques Used: Western Blot, Expressing, Negative Control, SDS Page

    Related Articles

    Western Blot:

    Article Title: Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor, et al. Targeting epidermal growth factor‐overexpressing triple‐negative breast cancer by natural killer cells expressing a specific chimeric antigen receptor
    Article Snippet: .. 2.5 Western blottingThe anti‐CD3ζ, anti‐EGFR, and anti‐β‐actin antibodies were purchased from Abcam, Cell Signal Technology, and Sigma, respectively. .. The anti‐rabbit and anti‐mouse secondary antibodies were purchased from Santa Cruz Biotechnology.

    Incubation:

    Article Title: CD60b: Enriching Neural Stem/Progenitor Cells from Rat Development into Adulthood
    Article Snippet: .. The sections were incubated with the primary antibody mouse IgM anti-CD60b (1 : 100, from Santa Cruz Biotechnology or Jones from Sigma, St. Louis, MO, USA) alone or in combination with one of the following primary antibodies or lectin: anti-nestin (1 : 100, Chemicon, Temecula, CA, USA), anti-vimentin (1 : 200, Chemicon), anti-GFAP (1 : 500, Dako, Denmark), anti-EGFR (1 : 250, Abcam, Cambridge, MA, USA), anti-doublecortin (1 : 500, Chemicon), PNA (Peanut agglutinin; 1 : 100, Sigma) overnight at 4°C. .. After washes with 0.01% Triton X-100 in PBS, sections were incubated with the secondary antibody goat anti-mouse IgM Cy3-conjugated (1 : 500, Jackson) alone or in combination with one of the following secondary antibodies: goat anti-rabbit Alexa Fluor 488-conjugated (1 : 500, Invitrogen) or goat anti-mouse IgG Alexa Fluor 488-conjugated (1 : 500, Invitrogen), for 2 h at room temperature.

    Article Title: Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts
    Article Snippet: .. After blocking with 5% bovine serum albumin at room temperature for one hour, primary antibodies including anti‐TS, anti‐EGFR, anti‐phospho‐EGFR, anti‐AKT, anti‐phospho‐AKT, anti‐MAPK, anti‐phospho‐MAPK, and anti‐β‐actin antibody (Abcam, Cambridge, MA, USA) were added overnight and incubated at 4°C. .. The membranes were washed three times the next day with tris‐buffered saline and tween‐20 before adding horseradish peroxidase‐conjugated secondary antibody (Proteintech Group Inc., Rosemont, IL, USA).

    Blocking Assay:

    Article Title: Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts
    Article Snippet: .. After blocking with 5% bovine serum albumin at room temperature for one hour, primary antibodies including anti‐TS, anti‐EGFR, anti‐phospho‐EGFR, anti‐AKT, anti‐phospho‐AKT, anti‐MAPK, anti‐phospho‐MAPK, and anti‐β‐actin antibody (Abcam, Cambridge, MA, USA) were added overnight and incubated at 4°C. .. The membranes were washed three times the next day with tris‐buffered saline and tween‐20 before adding horseradish peroxidase‐conjugated secondary antibody (Proteintech Group Inc., Rosemont, IL, USA).

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    Extracellular rhHSP70 induces a <t>TLR4-dependent</t> <t>EGFR</t> transactivation leading to the GJIC abrogation A. Western blot analysis of EGFR Tyr-1068 phosphorylation and TLR4 expression in HMEC, unstimulated (control) or stimulated with rhHSP70 or LPS (1 μg/ml) for 15 min. When indicated, cells were pre-treated for 60 min with polymyxin B (PMB10 μM) or the neutralysing anti-TLR4 ( Ab TLR4 10 μg/ml). Lower panel shows changes in the band intensity (mean ± SD, n=3; Hsc70 as loading control). B. Tyrosine phosphorylation of EGFR by rhHSP70 involves the kinase JAK2. Western blot analysis of EGFR phosphotyrosine (P-Tyr) after EGFR immunoprecipitation in HMEC. Cell pretreatment with the kinase inhibitors AG1478 (AG14; 5μM), CGP77675 (CGP; 1μM), AG490 (50μM) for 30 min before exposure to rhHSP70 or 100 ng/ml EGF for 15 min. A boiled rhHSP70 (100°C, 30 min) known to denaturize protein but not LPS, was used to evaluate the contribution of contaminants to the EGFR activation. Lower panel shows changes in band intensity (mean ± SD, n=5; **P
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    Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the <t>B8-mCherry</t> fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and <t>EGFR</t> expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.
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    Expression of thymidylate synthase (TS) and related <t>EGFR</t> signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, <t>AKT,</t> and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P
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    Effects of the upregulation or downregulation of <t>RBM10</t> expression on <t>EGFR</t> expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P
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    Image Search Results


    Extracellular rhHSP70 induces a TLR4-dependent EGFR transactivation leading to the GJIC abrogation A. Western blot analysis of EGFR Tyr-1068 phosphorylation and TLR4 expression in HMEC, unstimulated (control) or stimulated with rhHSP70 or LPS (1 μg/ml) for 15 min. When indicated, cells were pre-treated for 60 min with polymyxin B (PMB10 μM) or the neutralysing anti-TLR4 ( Ab TLR4 10 μg/ml). Lower panel shows changes in the band intensity (mean ± SD, n=3; Hsc70 as loading control). B. Tyrosine phosphorylation of EGFR by rhHSP70 involves the kinase JAK2. Western blot analysis of EGFR phosphotyrosine (P-Tyr) after EGFR immunoprecipitation in HMEC. Cell pretreatment with the kinase inhibitors AG1478 (AG14; 5μM), CGP77675 (CGP; 1μM), AG490 (50μM) for 30 min before exposure to rhHSP70 or 100 ng/ml EGF for 15 min. A boiled rhHSP70 (100°C, 30 min) known to denaturize protein but not LPS, was used to evaluate the contribution of contaminants to the EGFR activation. Lower panel shows changes in band intensity (mean ± SD, n=5; **P

    Journal: Oncotarget

    Article Title: Oncogenic extracellular HSP70 disrupts the gap-junctional coupling between capillary cells

    doi:

    Figure Lengend Snippet: Extracellular rhHSP70 induces a TLR4-dependent EGFR transactivation leading to the GJIC abrogation A. Western blot analysis of EGFR Tyr-1068 phosphorylation and TLR4 expression in HMEC, unstimulated (control) or stimulated with rhHSP70 or LPS (1 μg/ml) for 15 min. When indicated, cells were pre-treated for 60 min with polymyxin B (PMB10 μM) or the neutralysing anti-TLR4 ( Ab TLR4 10 μg/ml). Lower panel shows changes in the band intensity (mean ± SD, n=3; Hsc70 as loading control). B. Tyrosine phosphorylation of EGFR by rhHSP70 involves the kinase JAK2. Western blot analysis of EGFR phosphotyrosine (P-Tyr) after EGFR immunoprecipitation in HMEC. Cell pretreatment with the kinase inhibitors AG1478 (AG14; 5μM), CGP77675 (CGP; 1μM), AG490 (50μM) for 30 min before exposure to rhHSP70 or 100 ng/ml EGF for 15 min. A boiled rhHSP70 (100°C, 30 min) known to denaturize protein but not LPS, was used to evaluate the contribution of contaminants to the EGFR activation. Lower panel shows changes in band intensity (mean ± SD, n=5; **P

    Article Snippet: Neutralizing anti-TLR4 and mouse anti-EGFR (ErB1) were from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing, Immunoprecipitation, Activation Assay

    Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the B8-mCherry fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and EGFR expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Cross-priming induces immunodomination in the presence of viral MHC class I inhibition

    doi: 10.1371/journal.ppat.1006883

    Figure Lengend Snippet: Secretion of the immunodominant antigen is not required for immunodominance. (A) Schematic representation of the B8-mCherry fusion proteins; the location of the signal peptide, GGSGGS linker, TMD, and mCherry are depicted. (B) B8TMmC is not secreted. HeLa cells were infected at an MOI of 5 with B8TMmC or B8mC. Cells and supernatant were harvested at 4 hpi for subcellular fractionation and mCherry and EGFR expression was determined by western blot; equal loading and transfer of samples was confirmed with ponceau S red (P-Red) staining. CE = cytoplasmic extract; ME = membrane extract; SN = supernatant. Data are representative of two independent experiments. (C, D) Comparable CTL priming by B8TMmC and B8mC. CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 5 x 10 3 pfu (C) and 1.5 x 10 4 pfu (D) B8TMmC or B8mC were determined by ex vivo restimulation with CPXV peptides and ICS at 8 dpi. Data are representative of two independent experiments. (E) Cell-associated antigen is cross-presented more efficiently than soluble antigen. B8-specific CD8 + T cell responses in the spleen of B6 (n = 5) i.n. infected with 1.5 x 10 5 pfu B8TMmC or B8mC were determined by tetramer staining at 8, 9, and 10 dpi. Data are representative of two independent experiments. (F) CD8 + T cell responses require BATF3 + DCs. B6 and Batf3 -/- mice (n = 7–10) were i.n. infected with 5 x 10 3 pfu B8TMmC or B8mC and the B8-specific CD8 + T cell responses in the spleen were determined at 6 dpi. n = 3 mock-infected mice. Data are the combined results of three independent experiments.

    Article Snippet: Immunoblotting was performed using rabbit polyclonal anti-mCherry and rabbit monoclonal anti-EGFR (Abcam) followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell Signalling).

    Techniques: Infection, Fractionation, Expressing, Western Blot, Staining, CTL Assay, Ex Vivo, Mouse Assay

    Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    Journal: Thoracic Cancer

    Article Title: Efficacy of combined icotinib and pemetrexed in EGFR mutant lung adenocarcinoma cell line xenografts

    doi: 10.1111/1759-7714.12818

    Figure Lengend Snippet: Expression of thymidylate synthase (TS) and related EGFR signaling pathway proteins in tumor xenografts after the administration of different treatments ( n = 6 mice/group). Groups: control; Ico, icotinib; Ico‐Pem, sequential Ico followed by Pem; Ico + Pem, concurrent Ico and Pem; Pem, pemetrexed; Pem‐Ico, sequential Pem followed by Ico. ( a ) The effects of different combinations of Ico and Pem on TS expression and EGFR, AKT, and MAPK phosphorylation in tumor tissues was detected by Western blotting. The relative ( b ) TS ( c ) phospho‐EGFR, ( d ) phospho‐AKT, and ( e ) phospho‐MAPK expression levels. Data are shown as the mean ± standard deviation of triplicate measurements. P

    Article Snippet: After blocking with 5% bovine serum albumin at room temperature for one hour, primary antibodies including anti‐TS, anti‐EGFR, anti‐phospho‐EGFR, anti‐AKT, anti‐phospho‐AKT, anti‐MAPK, anti‐phospho‐MAPK, and anti‐β‐actin antibody (Abcam, Cambridge, MA, USA) were added overnight and incubated at 4°C.

    Techniques: Expressing, Mouse Assay, End-sequence Profiling, Western Blot, Standard Deviation

    Effects of the upregulation or downregulation of RBM10 expression on EGFR expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P

    Journal: International Journal of Oncology

    Article Title: Functional role of RBM10 in lung adenocarcinoma proliferation

    doi: 10.3892/ijo.2018.4643

    Figure Lengend Snippet: Effects of the upregulation or downregulation of RBM10 expression on EGFR expression. (A) Effects of RBM10 expression on EGFR expression in A549 cells examined by immunofluorescence assay; magnification, ×400. (B) Effects of RBM10 expression on EGFR expression in H1299 cells examined by immunofluorescence assay; magnification, ×400. (C) Effects of RBM10 expression on EGFR expression in A549 cells examined by western blot analysis and quantitative analysis of the western blots. (D) Effect of RBM10 expression on EGFR expression in H1299 cells examined by western blot analysis and quantitative analysis of the western blots. # P

    Article Snippet: The antibodies used were as follows: Rabbit anti-RBM10 (ab72423, 1:2,000 dilution), rabbit anti-p53 (ab131442, 1:500 dilution), rabbit anti-EGFR (ab131498, 1:500 dilution), rabbit anti-Bax (ab182733, 1:1,000 dilution), rabbit anti-Bcl-2 (ab32124, 1:1,000 dilution), rabbit anti-caspase-8 (ab25901, 1:1,000 dilution) and rabbit anti-GAPDH (ab37168, 1:1,000 dilution) antibodies were obtained from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Immunofluorescence, Western Blot