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Inhibition of <t>EGFR</t> pathway blocked <t>ERK/AKT/cyclin</t> D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2
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1) Product Images from "Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human Keratinocytes"

Article Title: Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human Keratinocytes

Journal: Photochemistry and photobiology

doi: 10.1111/j.1751-1097.2008.00531.x

Inhibition of EGFR pathway blocked ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2
Figure Legend Snippet: Inhibition of EGFR pathway blocked ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2

Techniques Used: Inhibition, Incubation

Cells were exposed to UVB as in . Cells were harvested at 1.5, 3 or 6 h following UVB radiation for Western blot analysis using specific antibodies against cyclin D1, p-AKT, AKT, p-ERK, ERK, p-EGFR, EGFR and beta-actin (an equal loading control)
Figure Legend Snippet: Cells were exposed to UVB as in . Cells were harvested at 1.5, 3 or 6 h following UVB radiation for Western blot analysis using specific antibodies against cyclin D1, p-AKT, AKT, p-ERK, ERK, p-EGFR, EGFR and beta-actin (an equal loading control)

Techniques Used: Western Blot

2) Product Images from "Nuclear EGFR Contributes to Acquired Resistance to Cetuximab"

Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

Journal: Oncogene

doi: 10.1038/onc.2009.234

Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P
Figure Legend Snippet: Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P

Techniques Used: Over Expression, In Vivo, Mouse Assay, Injection, Plasmid Preparation

Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.
Figure Legend Snippet: Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.

Techniques Used: Translocation Assay

Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P
Figure Legend Snippet: Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P

Techniques Used: Translocation Assay, SDS Page, Expressing, Clone Assay, Software, Flow Cytometry, Cytometry, Labeling

EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P
Figure Legend Snippet: EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P

Techniques Used: Sequencing, In Vitro, Construct, Infection, Clone Assay, Plasmid Preparation, Immunoprecipitation, SDS Page, Immunofluorescence, Staining, Expressing, Software, Proliferation Assay

3) Product Images from "WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression"

Article Title: WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression

Journal: Oncotarget

doi:

EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P
Figure Legend Snippet: EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P

Techniques Used: Expressing, Transfection, Incubation, Western Blot

WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P
Figure Legend Snippet: WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P

Techniques Used: Expressing, In Vivo, Stable Transfection, shRNA, Construct, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Migration, Mouse Assay, Injection, Staining, Imaging

4) Product Images from "CD73 is associated with poor prognosis in HNSCC"

Article Title: CD73 is associated with poor prognosis in HNSCC

Journal: Oncotarget

doi: 10.18632/oncotarget.11435

EGFR and adenosine signaling pathways are involved in CD73-dependent EMT in HNSCC cells ( A ) CAL27 cells were treated with siRNA targeting CD73 or control siRNA followed by incubation with MRS3588 for another 36 h. E-cadherin, N-cadherin, Vimentin and Slug levels were evaluated. GAPDH was the internal standard for protein loading. One-way ANOVA with post-Dunnett analysis was performed using GraphPad Prism5.* P
Figure Legend Snippet: EGFR and adenosine signaling pathways are involved in CD73-dependent EMT in HNSCC cells ( A ) CAL27 cells were treated with siRNA targeting CD73 or control siRNA followed by incubation with MRS3588 for another 36 h. E-cadherin, N-cadherin, Vimentin and Slug levels were evaluated. GAPDH was the internal standard for protein loading. One-way ANOVA with post-Dunnett analysis was performed using GraphPad Prism5.* P

Techniques Used: Incubation

5) Product Images from "The long non-coding RNA, GAS5, enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFR via downregulation of the IGF-1R expression"

Article Title: The long non-coding RNA, GAS5, enhances gefitinib-induced cell death in innate EGFR tyrosine kinase inhibitor-resistant lung adenocarcinoma cells with wide-type EGFR via downregulation of the IGF-1R expression

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-015-0140-6

Effect of GAS5 and gefitinib on the activity of the EGFR/PI3K/AKT pathway and IGF-1R in vivo . (A) Significant suppression of EGFR, downstream signaling molecules, and IGF-1R are seen following co-treatment with GAS5 and gefitinib. (B) Relative levels of P-EGFR, P-AKT, P-ERK, and P-IGF-1R proteins expression in four groups. The data represent the mean ± SD from three independent experiments. * P
Figure Legend Snippet: Effect of GAS5 and gefitinib on the activity of the EGFR/PI3K/AKT pathway and IGF-1R in vivo . (A) Significant suppression of EGFR, downstream signaling molecules, and IGF-1R are seen following co-treatment with GAS5 and gefitinib. (B) Relative levels of P-EGFR, P-AKT, P-ERK, and P-IGF-1R proteins expression in four groups. The data represent the mean ± SD from three independent experiments. * P

Techniques Used: Activity Assay, In Vivo, Expressing

Effect of GAS5 and gefitinib on the activity of the EGFR/PI3K/AKT pathway and IGF-1R in A549 cells. (A) Significant suppression of EGFR and downstream signaling molecules are seen following co-treatment with GAS5 and gefitinib. Dose-dependent downregulation of IGF-1R protein was also seen. (B) Relative levels of P-EGFR, P-AKT, P-ERK and P-IGF-1R proteins expression in six groups. The data represent the mean ± SD from three independent experiments. * P
Figure Legend Snippet: Effect of GAS5 and gefitinib on the activity of the EGFR/PI3K/AKT pathway and IGF-1R in A549 cells. (A) Significant suppression of EGFR and downstream signaling molecules are seen following co-treatment with GAS5 and gefitinib. Dose-dependent downregulation of IGF-1R protein was also seen. (B) Relative levels of P-EGFR, P-AKT, P-ERK and P-IGF-1R proteins expression in six groups. The data represent the mean ± SD from three independent experiments. * P

Techniques Used: Activity Assay, Expressing

6) Product Images from "Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells"

Article Title: Pigment Epithelium-Derived Factor (PEDF) Expression Induced by EGFRvIII Promotes Self-renewal and Tumor Progression of Glioma Stem Cells

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002152

EGFRvIII expression maintains stemness of GSCs. (A) Semiquantitative RT-PCR of EGFR-WT and EGFRvIII in various GSCs (EGFRvIII positive cells; CSC2, X01, X03, X04, X06, X08, X09, MD30, 1123NS, 83NS, and EGFRvIII negative cells; X02, Ex Vivo, and 528NS) and EGFRvIII-overexpressing Astrocyte (Astrocyte-EGFRvIII is used as EGFRvIII positive control). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Immunoblot (IB) analysis of EGFR, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 and X01 cells (day 0) and 10% serum-cultured CSC2 and X01 cells. Serum-cultured CSC2 and X01 cells were harvested after indicated time (days 4, 7, and 9). α-tubulin was used as a loading control. (C, D) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 cells (day 0) and 10% serum-cultured CSC2 cells (day 7) (C) and in serum-free GSC cultured X01 cells (day 0) and 10% serum-cultured X01 cells (day 7) (D). (E) Immunocytochemistry (ICC) of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 and X01 cells that were incubated in serum-free (day 0) or serum medium for 7 d (day 7). Nuclei were counterstained with DAPI (blue). (F) IB analysis of phosphorylated EGFR (p-EGFR), EGFR, Sox2, Nestin, and GFAP in CSC2 cells transfected with EGFRvIII small interfering RNA (siRNA) or its control. α-tubulin was used as a loading control. (G) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. GAPDH was used as a loading control. (H) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. Nuclei were counterstained with DAPI (blue). (I) Limiting dilution assay (LDA) was performed in CSC2 cells transfected with EGFRvIII siRNA or its control. p = 0.00966. (J) IB analysis of p-EGFR, EGFR, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. α-tubulin was used as a loading control. (K) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. GAPDH was used as a loading control. (L) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. Nuclei were counterstained with DAPI (blue). (M) LDA was performed in X02 infected with EGFRvIII-expressing lentiviral or control construct. p = 0.0000266.
Figure Legend Snippet: EGFRvIII expression maintains stemness of GSCs. (A) Semiquantitative RT-PCR of EGFR-WT and EGFRvIII in various GSCs (EGFRvIII positive cells; CSC2, X01, X03, X04, X06, X08, X09, MD30, 1123NS, 83NS, and EGFRvIII negative cells; X02, Ex Vivo, and 528NS) and EGFRvIII-overexpressing Astrocyte (Astrocyte-EGFRvIII is used as EGFRvIII positive control). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Immunoblot (IB) analysis of EGFR, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 and X01 cells (day 0) and 10% serum-cultured CSC2 and X01 cells. Serum-cultured CSC2 and X01 cells were harvested after indicated time (days 4, 7, and 9). α-tubulin was used as a loading control. (C, D) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in serum-free GSC cultured CSC2 cells (day 0) and 10% serum-cultured CSC2 cells (day 7) (C) and in serum-free GSC cultured X01 cells (day 0) and 10% serum-cultured X01 cells (day 7) (D). (E) Immunocytochemistry (ICC) of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 and X01 cells that were incubated in serum-free (day 0) or serum medium for 7 d (day 7). Nuclei were counterstained with DAPI (blue). (F) IB analysis of phosphorylated EGFR (p-EGFR), EGFR, Sox2, Nestin, and GFAP in CSC2 cells transfected with EGFRvIII small interfering RNA (siRNA) or its control. α-tubulin was used as a loading control. (G) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. GAPDH was used as a loading control. (H) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in CSC2 transfected with siEGFRvIII or siControl. Nuclei were counterstained with DAPI (blue). (I) Limiting dilution assay (LDA) was performed in CSC2 cells transfected with EGFRvIII siRNA or its control. p = 0.00966. (J) IB analysis of p-EGFR, EGFR, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. α-tubulin was used as a loading control. (K) Semiquantitative RT-PCR of EGFRvIII, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. GAPDH was used as a loading control. (L) ICC of EGFRvIII, p-STAT3, Sox2, Nestin, and GFAP in X02 infected with EGFRvIII-expressing lentiviral or control construct. Nuclei were counterstained with DAPI (blue). (M) LDA was performed in X02 infected with EGFRvIII-expressing lentiviral or control construct. p = 0.0000266.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Ex Vivo, Positive Control, Cell Culture, Immunocytochemistry, Incubation, Transfection, Small Interfering RNA, Limiting Dilution Assay, Infection, Construct

EGFRvIII/STAT3/PEDF signaling in GSCs. (A) IB analysis of EGFR, EGFRvIII, p-EGFR, p-STAT3, STAT3, and PEDF (in medium) in 13 GSCs. α-tubulin was used as a loading control. EGFRvIII + /PEDF high GSCs are CSC2, X01, X03, X04, X06, X08, and X09 cells. EGFRvIII + /PEDF low GSCs are MD30, 1123NS, and 83NS cells. EGFRvIII - /PEDF low GSCs are X02, Ex Vivo, and 528NS cells. (B) Histopathology of Balb-c/nu mouse brain tissue was orthotopically injected with three representative types of GSCs (A). Upper panel is hematoxylin and eosin (H E) staining of the whole brain. Red box indicates a site of corpus callosum far from the injection site. This site was stained by H E, Nestin, EGFRvIII, p-STAT3, and Sox2.
Figure Legend Snippet: EGFRvIII/STAT3/PEDF signaling in GSCs. (A) IB analysis of EGFR, EGFRvIII, p-EGFR, p-STAT3, STAT3, and PEDF (in medium) in 13 GSCs. α-tubulin was used as a loading control. EGFRvIII + /PEDF high GSCs are CSC2, X01, X03, X04, X06, X08, and X09 cells. EGFRvIII + /PEDF low GSCs are MD30, 1123NS, and 83NS cells. EGFRvIII - /PEDF low GSCs are X02, Ex Vivo, and 528NS cells. (B) Histopathology of Balb-c/nu mouse brain tissue was orthotopically injected with three representative types of GSCs (A). Upper panel is hematoxylin and eosin (H E) staining of the whole brain. Red box indicates a site of corpus callosum far from the injection site. This site was stained by H E, Nestin, EGFRvIII, p-STAT3, and Sox2.

Techniques Used: Ex Vivo, Histopathology, Injection, Staining

PEDF expression maintains stemness and self-renewal of GSCs. (A, C) LDA was performed in GSCs (CSC2 and X01) infected with shPEDF1-expressing lentiviral, shPEDF1 with PEDF-overexpressing lentiviral, or control construct. CSC2-Con or CSC-shPEDF1; p = 0.000201, CSC2-Con or CSC-shPEDF1-PEDF; p = 0.576, CSC2-shPEDF1 or CSC-shPEDF1-PEDF; p = 2.23e-05 (A) and X01-Con or X01-shPEDF1; p = 0.000265, X01-Con or X01-shPEDF1-PEDF; p = 0.589, X01-shPEDF1 or X01-shPEDF1-PEDF; p = 2.99e-05 (C). (B, D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in CSC2-Con, CSC2-shPEDF1, or CSC2-shPEDF1-PEDF (B) and X01-Con, X01-shPEDF1, or X01-shPEDF1-PEDF (D). β-actin was used as a loading control. (E) LDA was performed in X02 infected with PEDF-expressing lentiviral or control construct. X02-Con or X02-PEDF; p = 0.00936. (F) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X02-Con or X02-PEDF cells. β-actin was used as a loading control. (G) IB analysis of PEDF (in medium), p-EGFR, EGFR, p-STAT3, STAT3, Nestin, Sox2, and GFAP in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. (H) Sphere formation assay was performed in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM ( n = 3). * p
Figure Legend Snippet: PEDF expression maintains stemness and self-renewal of GSCs. (A, C) LDA was performed in GSCs (CSC2 and X01) infected with shPEDF1-expressing lentiviral, shPEDF1 with PEDF-overexpressing lentiviral, or control construct. CSC2-Con or CSC-shPEDF1; p = 0.000201, CSC2-Con or CSC-shPEDF1-PEDF; p = 0.576, CSC2-shPEDF1 or CSC-shPEDF1-PEDF; p = 2.23e-05 (A) and X01-Con or X01-shPEDF1; p = 0.000265, X01-Con or X01-shPEDF1-PEDF; p = 0.589, X01-shPEDF1 or X01-shPEDF1-PEDF; p = 2.99e-05 (C). (B, D) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in CSC2-Con, CSC2-shPEDF1, or CSC2-shPEDF1-PEDF (B) and X01-Con, X01-shPEDF1, or X01-shPEDF1-PEDF (D). β-actin was used as a loading control. (E) LDA was performed in X02 infected with PEDF-expressing lentiviral or control construct. X02-Con or X02-PEDF; p = 0.00936. (F) IB analysis of PEDF (in medium), Sox2, Nestin, and GFAP in X02-Con or X02-PEDF cells. β-actin was used as a loading control. (G) IB analysis of PEDF (in medium), p-EGFR, EGFR, p-STAT3, STAT3, Nestin, Sox2, and GFAP in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. (H) Sphere formation assay was performed in X02-Con, X02-EGFRvIII, X02-shPEDF2, or X02-EGFRvIII coinfected with shPEDF2-expressing lentiviral construct. The graph represents the average proportion of sphere number. Counted sphere size is greater than 100 μm. All error bars represent mean ± SEM ( n = 3). * p

Techniques Used: Expressing, Infection, Construct, Tube Formation Assay

7) Product Images from "Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial-mesenchymal transition of breast cancer cells"

Article Title: Involvement of NF-κB/miR-448 regulatory feedback loop in chemotherapy-induced epithelial-mesenchymal transition of breast cancer cells

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2010.103

Identification of AR–EGFR axis as downstream pathway of SATB1. ( a ) Adriamycin-treated MCF7 cells were processed for ChIP using an antibody against SATB1. DNA was interrogated with primers specific for AR promoter. ‘−' and ‘+'
Figure Legend Snippet: Identification of AR–EGFR axis as downstream pathway of SATB1. ( a ) Adriamycin-treated MCF7 cells were processed for ChIP using an antibody against SATB1. DNA was interrogated with primers specific for AR promoter. ‘−' and ‘+'

Techniques Used: Chromatin Immunoprecipitation

8) Product Images from "RAF1-MEK1-ERK/AKT axis may confer NSCLC cell lines resistance to erlotinib"

Article Title: RAF1-MEK1-ERK/AKT axis may confer NSCLC cell lines resistance to erlotinib

Journal: International Journal of Clinical and Experimental Pathology

doi:

The activation status of EGFR/ERK/AKT in 3 NSCLC cell lines. U1752, Calu-6 and NCI-H292 cells were applied to western blotting for EGFR, p-EGFR (Y1173), p-EGFR (Y1086), ERK, p-ERK (T202/204), AKT, p-AKT (S473). GAPDH was used as input control.
Figure Legend Snippet: The activation status of EGFR/ERK/AKT in 3 NSCLC cell lines. U1752, Calu-6 and NCI-H292 cells were applied to western blotting for EGFR, p-EGFR (Y1173), p-EGFR (Y1086), ERK, p-ERK (T202/204), AKT, p-AKT (S473). GAPDH was used as input control.

Techniques Used: Activation Assay, Western Blot

9) Product Images from "Sensitization of cerebral tissue in nude mice with photodynamic therapy induces ADAM17/TACE and promotes glioma cell invasion"

Article Title: Sensitization of cerebral tissue in nude mice with photodynamic therapy induces ADAM17/TACE and promotes glioma cell invasion

Journal: Cancer letters

doi: 10.1016/j.canlet.2008.02.023

ADAM17, EGFR, p-EGFR, Akt, p-Akt expression in normal brain. The positive ADAM17-immunoreactive area within the area of PDT treatment and contralateral were analyzed by MCID. 80J/cm2 PDT significantly increased ADAM17 positive area (10.89%) compared to
Figure Legend Snippet: ADAM17, EGFR, p-EGFR, Akt, p-Akt expression in normal brain. The positive ADAM17-immunoreactive area within the area of PDT treatment and contralateral were analyzed by MCID. 80J/cm2 PDT significantly increased ADAM17 positive area (10.89%) compared to

Techniques Used: Expressing

ADAM17, EGFR, p-EGFR, Akt, p-Akt expression in tumors implanted in nude mice. The positive ADAM17, EGFR, pEGFR, Akt and p-Akt-immunoreactive area within tumors implanted in nude mice with or without pro-PDT treatment were analyzed. Expression of ADAM17,
Figure Legend Snippet: ADAM17, EGFR, p-EGFR, Akt, p-Akt expression in tumors implanted in nude mice. The positive ADAM17, EGFR, pEGFR, Akt and p-Akt-immunoreactive area within tumors implanted in nude mice with or without pro-PDT treatment were analyzed. Expression of ADAM17,

Techniques Used: Expressing, Mouse Assay

10) Product Images from "CANNABINOID RECEPTOR-2 AGONIST INHIBITS MACROPHAGE INDUCED EMT IN NON-SMALL CELL LUNG CANCER BY DOWNREGULATION OF EGFR PATHWAY"

Article Title: CANNABINOID RECEPTOR-2 AGONIST INHIBITS MACROPHAGE INDUCED EMT IN NON-SMALL CELL LUNG CANCER BY DOWNREGULATION OF EGFR PATHWAY

Journal: Molecular carcinogenesis

doi: 10.1002/mc.22451

JWH-015 inhibits EGF induced proliferation in NSCLC cells. (A) NSCLC cell lines- A549, CALU1 and ED1 were subjected to immunoblot to determine expression of CB2 and EGFR. GAPDH is loading control. A549 (B), CALU1 (C) and EDI (D) cells were serum starved and treated with control, JWH-015 (5μM) in absence or presence of EGF (100ng/ml) for 48h and analyzed for viability by MTT assay. P
Figure Legend Snippet: JWH-015 inhibits EGF induced proliferation in NSCLC cells. (A) NSCLC cell lines- A549, CALU1 and ED1 were subjected to immunoblot to determine expression of CB2 and EGFR. GAPDH is loading control. A549 (B), CALU1 (C) and EDI (D) cells were serum starved and treated with control, JWH-015 (5μM) in absence or presence of EGF (100ng/ml) for 48h and analyzed for viability by MTT assay. P

Techniques Used: Expressing, MTT Assay

JWH-015 inhibits M2 macrophage induced EMT in A549 cells. (A) 1000 individual A549 cells were subjected to colony formation assay by treating with control or JWH-015 (5μM) in the presence of M2 CM for six days. Colonies were stained and counted. A549 cells were treated with control or JWH-015 (5μM) for 48h and subjected to M2-polarized TAM CM-induced migration (B) and invasion (C). Number of cells migrated or invaded were stained and counted. (D) A549 cells were treated with control or JWH-015 (5μM) for 24h in the presence of M2 CM, then conditioned media was replaced by fresh media for another 48h and the supernatants were concentrated and run on zymogram gels. A549 cells were pre-treated with control or JWH-015 (5μM) for 24h, stimulated with M2 CM for 48h and subjected to Immunoblot to determine expression of P-FAK and VCAM-1 (left panel) with quantification (right panel) (E), EGFR signaling pathway like P-STAT3, P-ERK (F) and EMT markers (left panel) with quantification (right panel) (G) and also subjected to Real time PCR (H). GAPDH is loading control. JWH-015 (JWH), M2-polarized TAM CM (M2 CM). P
Figure Legend Snippet: JWH-015 inhibits M2 macrophage induced EMT in A549 cells. (A) 1000 individual A549 cells were subjected to colony formation assay by treating with control or JWH-015 (5μM) in the presence of M2 CM for six days. Colonies were stained and counted. A549 cells were treated with control or JWH-015 (5μM) for 48h and subjected to M2-polarized TAM CM-induced migration (B) and invasion (C). Number of cells migrated or invaded were stained and counted. (D) A549 cells were treated with control or JWH-015 (5μM) for 24h in the presence of M2 CM, then conditioned media was replaced by fresh media for another 48h and the supernatants were concentrated and run on zymogram gels. A549 cells were pre-treated with control or JWH-015 (5μM) for 24h, stimulated with M2 CM for 48h and subjected to Immunoblot to determine expression of P-FAK and VCAM-1 (left panel) with quantification (right panel) (E), EGFR signaling pathway like P-STAT3, P-ERK (F) and EMT markers (left panel) with quantification (right panel) (G) and also subjected to Real time PCR (H). GAPDH is loading control. JWH-015 (JWH), M2-polarized TAM CM (M2 CM). P

Techniques Used: Colony Assay, Staining, Migration, Expressing, Real-time Polymerase Chain Reaction

JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of tumor cells by downregulation of EGFR signaling. (A) Xenograft tumors (n-5) were subjected to flow analysis. F4/80 cells isolated were gated to check for CD11b+CD206+ cells to determine the population of M2 macrophages. (B) Representative photomicrographs of Arginase-1 and N-cadherin staining of tumors extracted from experimental groups. (C) Xenograft tumors (n-5) were subjected to Real Time PCR to determine expression of EMT markers. (D) Xenograft tumors (n-5) isolated were subjected to Western blot to determine expression of EGFR signaling targets. GAPDH is loading control. (E) Xenograft tumors (n-5) were subjected to Real Time PCR to determine expression of MMP2. P
Figure Legend Snippet: JWH-015 decreases macrophage recruitment to tumor site and inhibits EMT of tumor cells by downregulation of EGFR signaling. (A) Xenograft tumors (n-5) were subjected to flow analysis. F4/80 cells isolated were gated to check for CD11b+CD206+ cells to determine the population of M2 macrophages. (B) Representative photomicrographs of Arginase-1 and N-cadherin staining of tumors extracted from experimental groups. (C) Xenograft tumors (n-5) were subjected to Real Time PCR to determine expression of EMT markers. (D) Xenograft tumors (n-5) isolated were subjected to Western blot to determine expression of EGFR signaling targets. GAPDH is loading control. (E) Xenograft tumors (n-5) were subjected to Real Time PCR to determine expression of MMP2. P

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

11) Product Images from "Autophagosome-Mediated EGFR Down-Regulation Induced by the CK2 Inhibitor Enhances the Efficacy of EGFR-TKI on EGFR-Mutant Lung Cancer Cells with Resistance by T790M"

Article Title: Autophagosome-Mediated EGFR Down-Regulation Induced by the CK2 Inhibitor Enhances the Efficacy of EGFR-TKI on EGFR-Mutant Lung Cancer Cells with Resistance by T790M

Journal: PLoS ONE

doi: 10.1371/journal.pone.0114000

The inhibition of CX-4945-induced autophagy led to decreased apoptosis. A, PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h and then were fixed with methanol, immunostained with anti-LC3 (red), anti-EGFR (green), and DAPI (blue), and analyzed by confocal microscopy to determine the intracellular localization of EGFR. B, The suppression of Atg7 by siRNA treatment was detected by Western blot analysis. PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h in the presence or absence of 3MA (2 mM) and Atg7 siRNA (100 nM). The modulation of EGFR was detected by Western blot analysis. C and D, Cells were treated with drugs as in Fig. 2 under the presence or absence of 3MA and Atg7 siRNA. Cleavage of PARP-1 and caspase-3 was shown by Western blot analysis. Apoptosis was assessed by Annexin V-FITC/Propidium iodide staining and flow cytometry. The results are representative of at least 3 independent experiments, and the error bars signify standard deviations (±SDs). *p
Figure Legend Snippet: The inhibition of CX-4945-induced autophagy led to decreased apoptosis. A, PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h and then were fixed with methanol, immunostained with anti-LC3 (red), anti-EGFR (green), and DAPI (blue), and analyzed by confocal microscopy to determine the intracellular localization of EGFR. B, The suppression of Atg7 by siRNA treatment was detected by Western blot analysis. PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h in the presence or absence of 3MA (2 mM) and Atg7 siRNA (100 nM). The modulation of EGFR was detected by Western blot analysis. C and D, Cells were treated with drugs as in Fig. 2 under the presence or absence of 3MA and Atg7 siRNA. Cleavage of PARP-1 and caspase-3 was shown by Western blot analysis. Apoptosis was assessed by Annexin V-FITC/Propidium iodide staining and flow cytometry. The results are representative of at least 3 independent experiments, and the error bars signify standard deviations (±SDs). *p

Techniques Used: Inhibition, Confocal Microscopy, Western Blot, Staining, Flow Cytometry, Cytometry

Down-regulation of CK2α by siRNA treatment induced the autophagy in EGFR-TKI-resistant PC-9 cells. A and B, Cells were transfected with control or CK2α siRNA (100 nM) for 48 h. Cell numbers were determined with an ADAM-MC automatic cell counter. Pictures showing the autophagic vacuole formation (AVOs) were taken at ×20 magnification. *p
Figure Legend Snippet: Down-regulation of CK2α by siRNA treatment induced the autophagy in EGFR-TKI-resistant PC-9 cells. A and B, Cells were transfected with control or CK2α siRNA (100 nM) for 48 h. Cell numbers were determined with an ADAM-MC automatic cell counter. Pictures showing the autophagic vacuole formation (AVOs) were taken at ×20 magnification. *p

Techniques Used: Transfection

12) Product Images from "Multiple receptor tyrosine kinases converge on microRNA-134 to control KRAS, STAT5B, and glioblastoma"

Article Title: Multiple receptor tyrosine kinases converge on microRNA-134 to control KRAS, STAT5B, and glioblastoma

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2013.196

RTKs regulate miR-134 expression via MAPK and KLF4. ( a ) GBM U87 and GSC 1228 cells were treated with inhibitors of PI3K, MAPK, and STAT3 and assessed for miR-134 expression by qRT-PCR. Only MAPK inhibition led to an upregulation of miR-134 expression in both cell lines. ( b ) KLF4 predicted binding sites in the putative miR-134 promoter. ( c ) Immunoblots showing the effect of MET inhibition and activation on KLF4 expression in GBM cells. ( d ) Immunoblots showing the downregulation of KLF4 expression after MAPK inhibition in GBM cells and GSCs. ( e ) Rescue experiments showing that siRNA-based KLF4 knockdown reverses the inhibitory effects on miR-134 expression of MET, EGFR, and PDGFR activations with their respective growth factor (GF) ligands in GBM cells (two left panels). Right panel shows immunoblots with KLF4 knockdown. ( f ) ChIP/qPCR showing the binding of KLF4 to two KLF4 binding sites (site 1 and site 2) in the putative miR-134 promoter in response to MET inhibition or activation with Crizotinib (criz) or HGF, respectively. Vimentin (Vim) was used as a positive control. Left panel shows ChIP/qPCR and right panel shows its quantification. * P
Figure Legend Snippet: RTKs regulate miR-134 expression via MAPK and KLF4. ( a ) GBM U87 and GSC 1228 cells were treated with inhibitors of PI3K, MAPK, and STAT3 and assessed for miR-134 expression by qRT-PCR. Only MAPK inhibition led to an upregulation of miR-134 expression in both cell lines. ( b ) KLF4 predicted binding sites in the putative miR-134 promoter. ( c ) Immunoblots showing the effect of MET inhibition and activation on KLF4 expression in GBM cells. ( d ) Immunoblots showing the downregulation of KLF4 expression after MAPK inhibition in GBM cells and GSCs. ( e ) Rescue experiments showing that siRNA-based KLF4 knockdown reverses the inhibitory effects on miR-134 expression of MET, EGFR, and PDGFR activations with their respective growth factor (GF) ligands in GBM cells (two left panels). Right panel shows immunoblots with KLF4 knockdown. ( f ) ChIP/qPCR showing the binding of KLF4 to two KLF4 binding sites (site 1 and site 2) in the putative miR-134 promoter in response to MET inhibition or activation with Crizotinib (criz) or HGF, respectively. Vimentin (Vim) was used as a positive control. Left panel shows ChIP/qPCR and right panel shows its quantification. * P

Techniques Used: Expressing, Quantitative RT-PCR, Inhibition, Binding Assay, Western Blot, Activation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

miR-134 expression is downregulated in GBM cells, GSCs, and human tumors, where it inversely correlates with RTK activation levels. ( a ) qRT-PCR of miR-134 in GBM cells and GSCs showing lower levels of expression as compared with normal human astrocytes. ( b ) Quantitative RT-PCR of miR-134 in GSCs showing lower levels of expression as compared with NSCs. ( c ) Quantitative RT-PCR of miR-134 in GBM surgical specimens (T) showing lower levels of miR-134 than in normal brain (N). ( d ) Immunoblots showing p-MET levels in the same GBM specimens and normal brains as in ( c ). ( e ) Quantitative RT-PCR of miR-134 in GBM surgical specimens (G) showing lower levels of miR-134 than in normal brains (N). ( f ) Immunoblots showing p-EGFR and p-PDGFR levels in the same GBM specimens and normal brains as in ( e ). ( g ) Correlations between miR-134 and p-MET (left panel), miR-134 and p-EGFR (middle panel), and miR-134 and p-PDGFR (right panel) levels in GBM and normal brain tissues from ( c )/( d ) and ( e )/( f ), respectively. Correlation coefficient R values between miR-134 and p-MET, p-EGFR, and p-PDGFR were R =0.875 ( P
Figure Legend Snippet: miR-134 expression is downregulated in GBM cells, GSCs, and human tumors, where it inversely correlates with RTK activation levels. ( a ) qRT-PCR of miR-134 in GBM cells and GSCs showing lower levels of expression as compared with normal human astrocytes. ( b ) Quantitative RT-PCR of miR-134 in GSCs showing lower levels of expression as compared with NSCs. ( c ) Quantitative RT-PCR of miR-134 in GBM surgical specimens (T) showing lower levels of miR-134 than in normal brain (N). ( d ) Immunoblots showing p-MET levels in the same GBM specimens and normal brains as in ( c ). ( e ) Quantitative RT-PCR of miR-134 in GBM surgical specimens (G) showing lower levels of miR-134 than in normal brains (N). ( f ) Immunoblots showing p-EGFR and p-PDGFR levels in the same GBM specimens and normal brains as in ( e ). ( g ) Correlations between miR-134 and p-MET (left panel), miR-134 and p-EGFR (middle panel), and miR-134 and p-PDGFR (right panel) levels in GBM and normal brain tissues from ( c )/( d ) and ( e )/( f ), respectively. Correlation coefficient R values between miR-134 and p-MET, p-EGFR, and p-PDGFR were R =0.875 ( P

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

13) Product Images from "Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes"

Article Title: Overexpression of EGFR in Head and Neck Squamous Cell Carcinoma Is Associated with Inactivation of SH3GL2 and CDC25A Genes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0063440

Immunocytochemical analysis of EGFR, SH3GL2, CDC25A and p-EGFR. over night subconfluent cover slip culture of Hep2 cell line was treated with 5-aza-dc for 72 hour and protein expression of the genes were analyzed by ICC after fixing the cells. EGFR and p-EGFR showed reduced expression in treated cells (b) compare to non treated cells (a). On the contrary, SH3GL2 and CDC25A showed upregulation of cytoplasmic/nuclear expression after aza treatment (b) compare to untreated cells (a). Scale bars in microphotograph represent 50 µm.
Figure Legend Snippet: Immunocytochemical analysis of EGFR, SH3GL2, CDC25A and p-EGFR. over night subconfluent cover slip culture of Hep2 cell line was treated with 5-aza-dc for 72 hour and protein expression of the genes were analyzed by ICC after fixing the cells. EGFR and p-EGFR showed reduced expression in treated cells (b) compare to non treated cells (a). On the contrary, SH3GL2 and CDC25A showed upregulation of cytoplasmic/nuclear expression after aza treatment (b) compare to untreated cells (a). Scale bars in microphotograph represent 50 µm.

Techniques Used: Expressing, Immunocytochemistry

SH3GL2 and CDC25A mediated EGFR homeostasis. a) EGFR was degraded due to upregulation of SH3GL2 and CDC25A by 5-aza-dc treatment. Hep2 cell line was incubated with 20 µm of 5-aza-dc up to 120 h. Cells were harvested after zero hour of treatment and then every 24 h interval. Equal amounts of protein were subjected to western blotting. The amount of EGFR protein decreased gradually and degradation was maximum after 120 hour. Similarly, the expression of SH3GL2 and CDC25A was gradually increased after treatment. b) The amount of proteins (normalized band OD) was plotted as a function of time of 5-aza-dc treatment. The intensity of the bands were determined by densitometry and normalized with tubulin. c) SCC084 cell line was treated with siRNA of CDC25A and SH3GL2. Protein expression of the genes were analysed by western blot. Expression of EGFR and phosphorylated EGFR were assayed during knock down either of SH3GL2 and CDC25A or of both. Both EGFR and p-EGFR level was up regulated due to reduction of SH3GL2 and CDC25A. d) The amount of proteins (normalized band OD) was plotted. The intensity of the bands were determined by densitometry and normalized with actin. The bar diagram showing the level of EGFR and p-EGFR up regulation during siRNA treatment of SH3GL2 and CDC25A.
Figure Legend Snippet: SH3GL2 and CDC25A mediated EGFR homeostasis. a) EGFR was degraded due to upregulation of SH3GL2 and CDC25A by 5-aza-dc treatment. Hep2 cell line was incubated with 20 µm of 5-aza-dc up to 120 h. Cells were harvested after zero hour of treatment and then every 24 h interval. Equal amounts of protein were subjected to western blotting. The amount of EGFR protein decreased gradually and degradation was maximum after 120 hour. Similarly, the expression of SH3GL2 and CDC25A was gradually increased after treatment. b) The amount of proteins (normalized band OD) was plotted as a function of time of 5-aza-dc treatment. The intensity of the bands were determined by densitometry and normalized with tubulin. c) SCC084 cell line was treated with siRNA of CDC25A and SH3GL2. Protein expression of the genes were analysed by western blot. Expression of EGFR and phosphorylated EGFR were assayed during knock down either of SH3GL2 and CDC25A or of both. Both EGFR and p-EGFR level was up regulated due to reduction of SH3GL2 and CDC25A. d) The amount of proteins (normalized band OD) was plotted. The intensity of the bands were determined by densitometry and normalized with actin. The bar diagram showing the level of EGFR and p-EGFR up regulation during siRNA treatment of SH3GL2 and CDC25A.

Techniques Used: Incubation, Western Blot, Expressing

Immunohistochemical analysis of SH3GL2, CDC25A and p-EGFR. a) Distinct cytoplasmic/membrane expression of SH3GL2 in the basal lining/parabasal cells of normal oral epithelium and primary HNSCC samples were seen. Spinus layer showed high expression of the gene. #403T and #5999T showed low expression, #4465T and #2333T showed intermediate/high expression expression level of SH3GL2. b) Differential nuclear and cytoplasmic expression of CDC25A was seen in basal/parabasal/spinus layer cells of normal oral epithelium and tumor samples. Low cytoplasmic and nuclear expression of CDC25A was evident in basal and parabasal cells of normal epithelium, but higher expression in spinus layer. #2333T and #5999T showed low expression, #4465T and #403T showed high/intermediate nuclear and cytoplasmic expression level of CDC25A. Arrows pointed to nuclear/cytoplasmic/membrane expression. c) Distinct cytoplasmic and membrane bound expression of p-EGFR was seen in normal oral epithelium. The expression was high in basal layer but gradually decreased in parabasal and spinus layer. Arrows pointed to cytoplasmic/membrane/nucleus expression. #2333T and #5999T showed high expression, #4465T and #403 showed reduced expression of p-EGFR. Magnification of tissue samples is 20X, and for inset in tissues magnification is 40X. Scale bars in tissue sections represent 100 µm.
Figure Legend Snippet: Immunohistochemical analysis of SH3GL2, CDC25A and p-EGFR. a) Distinct cytoplasmic/membrane expression of SH3GL2 in the basal lining/parabasal cells of normal oral epithelium and primary HNSCC samples were seen. Spinus layer showed high expression of the gene. #403T and #5999T showed low expression, #4465T and #2333T showed intermediate/high expression expression level of SH3GL2. b) Differential nuclear and cytoplasmic expression of CDC25A was seen in basal/parabasal/spinus layer cells of normal oral epithelium and tumor samples. Low cytoplasmic and nuclear expression of CDC25A was evident in basal and parabasal cells of normal epithelium, but higher expression in spinus layer. #2333T and #5999T showed low expression, #4465T and #403T showed high/intermediate nuclear and cytoplasmic expression level of CDC25A. Arrows pointed to nuclear/cytoplasmic/membrane expression. c) Distinct cytoplasmic and membrane bound expression of p-EGFR was seen in normal oral epithelium. The expression was high in basal layer but gradually decreased in parabasal and spinus layer. Arrows pointed to cytoplasmic/membrane/nucleus expression. #2333T and #5999T showed high expression, #4465T and #403 showed reduced expression of p-EGFR. Magnification of tissue samples is 20X, and for inset in tissues magnification is 40X. Scale bars in tissue sections represent 100 µm.

Techniques Used: Immunohistochemistry, Expressing

Kaplan–Meier analysis of survival (up to 5 years) of HNSCC patients. a) Co-alteration of CDC25A and SH3GL2 was significantly associated with poor overall survival (OS). b) The significant association with poor overall survival of patients having co-alterations of the genes was also seen in presence of HPV infection; however, co-alterations did not associate significantly with OS in absence of HPV infection (c). d) Poor survival was also seen of the patients having high EGFR expression irrespective of HPV infection. d) Similarly, reduced SH3GL2 expression and high EGFR expression was a predictor for poor OS. However f), protein expression of p-EGFR and CDC25A did not show any significant association with survival of the patients. Survival time was defined as the time from the date of surgery to the date of last follow-up, known recurrence or death (up to 5 years). n, total number of samples; C+/−, CDC25A deletion present/absent; S+/−, SH3GL2 alterations present/absent; H+/−, HPV infection present/absent; EH/ML, EGFR protein expression high/medium to low; S-L/MH, SH3GL2 protein expression low/medium to high; C-H/ML, CDC25A protein expression high/medium to low; p-EH/LM, p-EGFR protein expression high/low to medium.
Figure Legend Snippet: Kaplan–Meier analysis of survival (up to 5 years) of HNSCC patients. a) Co-alteration of CDC25A and SH3GL2 was significantly associated with poor overall survival (OS). b) The significant association with poor overall survival of patients having co-alterations of the genes was also seen in presence of HPV infection; however, co-alterations did not associate significantly with OS in absence of HPV infection (c). d) Poor survival was also seen of the patients having high EGFR expression irrespective of HPV infection. d) Similarly, reduced SH3GL2 expression and high EGFR expression was a predictor for poor OS. However f), protein expression of p-EGFR and CDC25A did not show any significant association with survival of the patients. Survival time was defined as the time from the date of surgery to the date of last follow-up, known recurrence or death (up to 5 years). n, total number of samples; C+/−, CDC25A deletion present/absent; S+/−, SH3GL2 alterations present/absent; H+/−, HPV infection present/absent; EH/ML, EGFR protein expression high/medium to low; S-L/MH, SH3GL2 protein expression low/medium to high; C-H/ML, CDC25A protein expression high/medium to low; p-EH/LM, p-EGFR protein expression high/low to medium.

Techniques Used: Infection, Expressing

14) Product Images from "Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line *"

Article Title: Taurocholate Induces Cyclooxygenase-2 Expression via the Sphingosine 1-phosphate Receptor 2 in a Human Cholangiocarcinoma Cell Line *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.668277

Proposed signaling pathways involved in TCA-induced COX-2 expression in human CCA cells. Activation of S1PR2 by TCA directly activates ERK1/2/AKt via activated G proteins or indirectly activates ERK1/2/Akt via EGFR. Activated ERK1/2/Akt further activate
Figure Legend Snippet: Proposed signaling pathways involved in TCA-induced COX-2 expression in human CCA cells. Activation of S1PR2 by TCA directly activates ERK1/2/AKt via activated G proteins or indirectly activates ERK1/2/Akt via EGFR. Activated ERK1/2/Akt further activate

Techniques Used: Expressing, Activation Assay

15) Product Images from "The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries"

Article Title: The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191418

Proposed mechanism of GPER-mediated signaling in porcine coronary artery tension regulation. Activation of GPER by agonist G-1 increases cAMP production and Gβγ release, Gβγ in turn activates tyrosine kinase Src, and then metalloproteinases which cleaves and releases HB-EGF from its precursors ProHB-EGF, and HB-EGF binds and activates EGFR. Active EGFR stimulates its downstream target ERK1/2 and thus leads to contraction.
Figure Legend Snippet: Proposed mechanism of GPER-mediated signaling in porcine coronary artery tension regulation. Activation of GPER by agonist G-1 increases cAMP production and Gβγ release, Gβγ in turn activates tyrosine kinase Src, and then metalloproteinases which cleaves and releases HB-EGF from its precursors ProHB-EGF, and HB-EGF binds and activates EGFR. Active EGFR stimulates its downstream target ERK1/2 and thus leads to contraction.

Techniques Used: Activation Assay

16) Product Images from "Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression"

Article Title: Praeruptorin B Mitigates the Metastatic Ability of Human Renal Carcinoma Cells through Targeting CTSC and CTSV Expression

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21082919

Pra-B suppressed epidermal growth factor receptor (EGFR)–MEK–ERK activation in 786-O and ACHN cells. ( A ) 786-O and ACHN cells were treated with various concentrations of Pra-B (0, 10, 20, and 30 μM) for 24 h, after which the cells were harvested to detect MAPKs-related proteins (p-ERK, t-ERK, p-JNK, t-JNK, p-p38, t-p38) and ( B ) the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK protein expression levels through immunoblotting. The histogram represents the densitometric analysis of protein expression. β-actin was used as the loading control. * p
Figure Legend Snippet: Pra-B suppressed epidermal growth factor receptor (EGFR)–MEK–ERK activation in 786-O and ACHN cells. ( A ) 786-O and ACHN cells were treated with various concentrations of Pra-B (0, 10, 20, and 30 μM) for 24 h, after which the cells were harvested to detect MAPKs-related proteins (p-ERK, t-ERK, p-JNK, t-JNK, p-p38, t-p38) and ( B ) the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK protein expression levels through immunoblotting. The histogram represents the densitometric analysis of protein expression. β-actin was used as the loading control. * p

Techniques Used: Activation Assay, Expressing

Pra-B attenuated epidermal growth factor-induced migration ability through the EGFR signaling pathway. The cells were pretreated with EGF (20 ng/mL) for 2 h and then incubated with various concentrations of Pra-B (0, 10, 20, and 30 μM) for 24 h. ( A ) Cell migration and invasion were measured using an in vitro migration and Matrigel-based invasion assay. Quantification of migrating cells presented in terms of percentage of control (0 μM) is shown as a histogram. ( B ) Cells were harvested to detect the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK, CTSC, and CTSV protein expression levels through immunoblotting. β-actin was used as the loading control. The expression of these proteins was detected by densitometry as an average relative ratio compared to β-actin from three different experiments. ** p
Figure Legend Snippet: Pra-B attenuated epidermal growth factor-induced migration ability through the EGFR signaling pathway. The cells were pretreated with EGF (20 ng/mL) for 2 h and then incubated with various concentrations of Pra-B (0, 10, 20, and 30 μM) for 24 h. ( A ) Cell migration and invasion were measured using an in vitro migration and Matrigel-based invasion assay. Quantification of migrating cells presented in terms of percentage of control (0 μM) is shown as a histogram. ( B ) Cells were harvested to detect the p-EGFR, t-EGFR, p-MEK, t-MEK, p-ERK, t-ERK, CTSC, and CTSV protein expression levels through immunoblotting. β-actin was used as the loading control. The expression of these proteins was detected by densitometry as an average relative ratio compared to β-actin from three different experiments. ** p

Techniques Used: Migration, Incubation, In Vitro, Invasion Assay, Expressing

Illustration of how Pra-B inhibits the migration and invasion of human RCC cells through suppressing EGFR–MEK–ERK activation depending on CTSC and CTSV expression.
Figure Legend Snippet: Illustration of how Pra-B inhibits the migration and invasion of human RCC cells through suppressing EGFR–MEK–ERK activation depending on CTSC and CTSV expression.

Techniques Used: Migration, Activation Assay, Expressing

17) Product Images from "PLAG Exerts Anti-Metastatic Effects by Interfering with Neutrophil Elastase/PAR2/EGFR Signaling in A549 Lung Cancer Orthotopic Model"

Article Title: PLAG Exerts Anti-Metastatic Effects by Interfering with Neutrophil Elastase/PAR2/EGFR Signaling in A549 Lung Cancer Orthotopic Model

Journal: Cancers

doi: 10.3390/cancers12030560

PAR2/EGFR-mediated expression of EMT-related genes; PLAG accelerates PAR2 degradation through ⍺-arrestin expression and assembly of ubiquitin ligase: C1) Tumor-infiltrating neutrophils secrete neutrophil elastase, which stimulates the PAR2 receptor. C2) Neutrophil elastase-stimulated PAR2 starts intracellular trafficking via assembly of βARR2 and clathrin (internalization complex). Internalized PAR2 leads to MMPs activation. C3-1) MMP cleavage of pre-HB-EGF and release of HB-EGF. C3-2) Internalization complex was detached from PAR2 and C4-2) PAR2 returns to the plasma membrane (re-sensitization). C4-1) Released HB-EGF stimulates EGFR and C5) EMT-related gene expression. EGFR transactivation and signaling induces several genes involved in EMT of cancer cells. Major metastatic activity of cancer cells can be achieved through modulation of EMT-related gene expression.
Figure Legend Snippet: PAR2/EGFR-mediated expression of EMT-related genes; PLAG accelerates PAR2 degradation through ⍺-arrestin expression and assembly of ubiquitin ligase: C1) Tumor-infiltrating neutrophils secrete neutrophil elastase, which stimulates the PAR2 receptor. C2) Neutrophil elastase-stimulated PAR2 starts intracellular trafficking via assembly of βARR2 and clathrin (internalization complex). Internalized PAR2 leads to MMPs activation. C3-1) MMP cleavage of pre-HB-EGF and release of HB-EGF. C3-2) Internalization complex was detached from PAR2 and C4-2) PAR2 returns to the plasma membrane (re-sensitization). C4-1) Released HB-EGF stimulates EGFR and C5) EMT-related gene expression. EGFR transactivation and signaling induces several genes involved in EMT of cancer cells. Major metastatic activity of cancer cells can be achieved through modulation of EMT-related gene expression.

Techniques Used: Expressing, Activation Assay, Activity Assay

PLAG inhibition of EGFR transactivation mediated by the neutrophil elastase/PAR2/βARR2 signaling pathway in A549 cells: ( a ) Inhibition of the EGFR signaling pathway and PAR2 degradation by PLAG treatment in neutrophil-stimulated A549 cells evaluated by Western blot; ( b ) Induction of ⍺ARR expression by PLAG treatment was confirmed by Western blotting; ( c ) Changes in complex formation between endocytosis-related proteins and PAR2 degradation following PLAG treatment; ( d ) Validation of the PAR2 ubiquitination-inducing effect of PLAG in neutrophil-activated A549 cells; ( e ) Lysosomal PAR2 degradation in PLAG-treated cells was verified by confocal microscopy; ( f ) Changes in HB-EGF secretion following PLAG treatment and neutrophil stimulation in A549 cells; ( g ) Phosphorylation of EGFR; ( h ) and expression of PAR2 in the lung tissues of A549-orthotopically implanted mice were prominently reduced by PLAG treatment.
Figure Legend Snippet: PLAG inhibition of EGFR transactivation mediated by the neutrophil elastase/PAR2/βARR2 signaling pathway in A549 cells: ( a ) Inhibition of the EGFR signaling pathway and PAR2 degradation by PLAG treatment in neutrophil-stimulated A549 cells evaluated by Western blot; ( b ) Induction of ⍺ARR expression by PLAG treatment was confirmed by Western blotting; ( c ) Changes in complex formation between endocytosis-related proteins and PAR2 degradation following PLAG treatment; ( d ) Validation of the PAR2 ubiquitination-inducing effect of PLAG in neutrophil-activated A549 cells; ( e ) Lysosomal PAR2 degradation in PLAG-treated cells was verified by confocal microscopy; ( f ) Changes in HB-EGF secretion following PLAG treatment and neutrophil stimulation in A549 cells; ( g ) Phosphorylation of EGFR; ( h ) and expression of PAR2 in the lung tissues of A549-orthotopically implanted mice were prominently reduced by PLAG treatment.

Techniques Used: Inhibition, Western Blot, Expressing, Confocal Microscopy, Mouse Assay

18) Product Images from "Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB"

Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

Journal: Nutrients

doi: 10.3390/nu10111621

Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.
Figure Legend Snippet: Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.

Techniques Used: Binding Assay, Activity Assay, Western Blot, Immunoprecipitation, Labeling

19) Product Images from "Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells"

Article Title: Epstein-Barr Virus encoded LMP1 regulates cyclin D1 promoter activity by nuclear EGFR and STAT3 in CNE1 cells

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/1756-9966-32-90

LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.
Figure Legend Snippet: LMP1 increased the binding ability of transcription factors EGFR and STAT3 to cyclin D1 promoter in vitro . (A) STAT3 binding activities within the cyclin D1 promoter were examined by EMSA. A biotin-labeled wild-type STAT3 oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type STAT3 (lane 4), unlabeled mutant STAT3 oligonucleotides (lane 5), or noncompetitive unlabeled NF-κB oligonucleotide (NS, lane 6). Biotin-labeled mutant STAT3 oligonucleotide probe was incubated with nuclear extracts of the indicated NPC cell lines (lanes 8–9). (B) Ten micrograms of nuclear extracts were pre-incubated with biotin-labeled STAT3 oligonucleotide probe in the presence of inhibitors directed against different phosphorylation sites of STAT3 (indicated above each lane). (C) The biotin-labeled wild-type EGFR oligonucleotide probe was incubated with nuclear extracts of CNE1 and CNE1-LMP1 cells in the presence of a 200-fold excess of unlabeled wild-type EGFR (lane 4), unlabeled mutant EGFR oligonucleotides (lane 6) or noncompetitive unlabeled NFκB oligonucleotide (NS, lane 7), and then EGFR DNA binding activities were examined by EMSA. (D-E) The nuclear extracts of CNE1 and CNE1-LMP1 cells were pre-incubated with biotin-labeled EGFR oligonucleotide probe in the presence of inhibitors AG1478, directed against phosphorylation of EGFR, or DNAzyme 1 (DZ1), targeting LMP1. RD: relative density.

Techniques Used: Binding Assay, In Vitro, Labeling, Incubation, Mutagenesis

LMP1 induced co-localization of EGFR and STAT3 in the nucleus. Endogenous association of EGFR (A) with STAT3 (B) in NPC cells without or with LMP1 expression. Equal amounts of fractionated cellular proteins were immunoprecipitated with an anti-EGFR or anti-STAT3 antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, STAT3, nucleolin, and α-tubulin are shown as loading and fractionation controls. N: nuclear fraction, C: cytosolic fraction, IB: immunoblot.
Figure Legend Snippet: LMP1 induced co-localization of EGFR and STAT3 in the nucleus. Endogenous association of EGFR (A) with STAT3 (B) in NPC cells without or with LMP1 expression. Equal amounts of fractionated cellular proteins were immunoprecipitated with an anti-EGFR or anti-STAT3 antibody and loaded for Western blotting. Input samples from equal amounts of proteins blotted for EGFR, STAT3, nucleolin, and α-tubulin are shown as loading and fractionation controls. N: nuclear fraction, C: cytosolic fraction, IB: immunoblot.

Techniques Used: Expressing, Immunoprecipitation, Western Blot, Fractionation

Identification of an EGFR and STAT3 response element in the cyclin D1 promoter. (A) Schematic diagram of mutant cyclin D1 promoter constructs are shown. The expansion for EGFR and STAT3 binding site illustrates the wild-type sequence and frames the nucleotides replaced by mutations. (B-C) Dual luciferase-reporter assays were performed in LMP1-negative and LMP-positive CNE1 cells after co-transfection of a wild type or mutant cyclin D1 promoter-reporter construct, plasmids expressing wild-type EGFR or STAT3, and a Renilla luciferase transfection control plasmid. The fold induction by EGFR and STAT3 is displayed as the ratio of promoter activity obtained with wild-type compared to the DNA-binding mutant. (mean ± SD, n = 3, * p
Figure Legend Snippet: Identification of an EGFR and STAT3 response element in the cyclin D1 promoter. (A) Schematic diagram of mutant cyclin D1 promoter constructs are shown. The expansion for EGFR and STAT3 binding site illustrates the wild-type sequence and frames the nucleotides replaced by mutations. (B-C) Dual luciferase-reporter assays were performed in LMP1-negative and LMP-positive CNE1 cells after co-transfection of a wild type or mutant cyclin D1 promoter-reporter construct, plasmids expressing wild-type EGFR or STAT3, and a Renilla luciferase transfection control plasmid. The fold induction by EGFR and STAT3 is displayed as the ratio of promoter activity obtained with wild-type compared to the DNA-binding mutant. (mean ± SD, n = 3, * p

Techniques Used: Mutagenesis, Construct, Binding Assay, Sequencing, Luciferase, Cotransfection, Expressing, Transfection, Plasmid Preparation, Activity Assay

Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, * p
Figure Legend Snippet: Inhibitors and dominant negative mutants targeting the EGFR and STAT3 pathways attenuated LMP1-augmented cyclin D1 promoter activity. (A-B) Stable expression of EGFR-DN and STAT3β inhibited the LMP1-increased activity of cyclin D1. The indicated NPC cell lines were transfected with a cyclin D1 promoter-reporter construct, a Renilla luciferase transfection control plasmid, and an EGFR-DN or STAT3-β expression plasmid. Twenty-four hrs. after transfection, the cells were treated with DNAzymes or a control oligo (2 μM) for 12 hrs. Cells were harvested at 36 hrs. after transfection and subjected to the luciferase assay. Firefly luciferase was measured and normalized to Renilla luciferase activity. The results were expressed as fold induction of the reporter activity in vector-transfected CNE1 cells, which was assigned a value of 1. (mean ± SD, n =3, * p

Techniques Used: Dominant Negative Mutation, Activity Assay, Expressing, Transfection, Construct, Luciferase, Plasmid Preparation

Cyclin D1 expression is reduced in CNE1-LMP1 cells after treatment with EGFR siRNA and STAT3 siRNA. (A) Dual luciferase-reporter assays were performed in CNE1-LMP1 cells after co-transfection with either control siRNA (siControl), EGFR siRNA (siEGFR), or STAT3 siRNA (siSTAT3) in addition to cyclin D1 promoter-reporter constructs and a Renilla luciferase transfection control plasmid. Firefly luciferase was measured and normalized to Renilla luciferase activity. The fold change in cyclin D1 expression by the indicated siRNA is displayed in each case. The control siRNA served as a non-targeting control. (mean ± SD, n =3, * p
Figure Legend Snippet: Cyclin D1 expression is reduced in CNE1-LMP1 cells after treatment with EGFR siRNA and STAT3 siRNA. (A) Dual luciferase-reporter assays were performed in CNE1-LMP1 cells after co-transfection with either control siRNA (siControl), EGFR siRNA (siEGFR), or STAT3 siRNA (siSTAT3) in addition to cyclin D1 promoter-reporter constructs and a Renilla luciferase transfection control plasmid. Firefly luciferase was measured and normalized to Renilla luciferase activity. The fold change in cyclin D1 expression by the indicated siRNA is displayed in each case. The control siRNA served as a non-targeting control. (mean ± SD, n =3, * p

Techniques Used: Expressing, Luciferase, Cotransfection, Construct, Transfection, Plasmid Preparation, Activity Assay

LMP1 affected the interaction of EGFR and STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-EGFR antibody (A) or anti-STAT3 antibody (B) and analyzed by Western blotting with a STAT3 and EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG). ®-actin were used as an internal control of Inuput. The bottom panels show the 50 μg of input materials. IP: immunoprecipitation, IB: immunoblot, kDa: kilodalton.
Figure Legend Snippet: LMP1 affected the interaction of EGFR and STAT3. Two mg of protein from cell lysates were immunoprecipitated with an anti-EGFR antibody (A) or anti-STAT3 antibody (B) and analyzed by Western blotting with a STAT3 and EGFR antibodies. Negative controls included immunoprecipitation with an unrelated antibody (IgG). ®-actin were used as an internal control of Inuput. The bottom panels show the 50 μg of input materials. IP: immunoprecipitation, IB: immunoblot, kDa: kilodalton.

Techniques Used: Immunoprecipitation, Western Blot

20) Product Images from "Traditional Chinese Medicine CFF‐1 induced cell growth inhibition, autophagy, and apoptosis via inhibiting EGFR‐related pathways in prostate cancer"

Article Title: Traditional Chinese Medicine CFF‐1 induced cell growth inhibition, autophagy, and apoptosis via inhibiting EGFR‐related pathways in prostate cancer

Journal: Cancer Medicine

doi: 10.1002/cam4.1419

Level changes of p‐ EGFR and EGFR in LNC aP and PC 3 cells treated with CFF ‐1 or EGF and co‐treated with CFF ‐1 and EGF . (A and B) LNC aP and PC 3 cells were incubated and treated with increasing doses of CFF ‐1 (0, 2, 5, 10 mg/mL) for 24 h as indicated in figures. And then, cells were harvested for Western blot assays to check the protein levels of p‐ EGFR (Tyr1173), EGFR , and β ‐Actin (loading control). (C and D) LNC aP and PC 3 cells were incubated and treated with 5 mg/mL of CFF ‐1 and then stimulated by different concentrations of EGF (20 μ g/mL, 30 μ g/mL) for 10 min as indicated in figures. Cells were harvested and lysed for Western blot assay to check the protein levels of p‐ EGFR (Tyr1173), EGFR and β ‐Actin (loading control). (E and F) LNC aP and PC 3 cells were pretreated with the indicated concentrations of CFF ‐1 (0, 5, 10 mg/mL) for 24 h and then stimulated by EGF (30 μ g/mL) for 10 min as indicated in figures. Cells were harvested for Western blot assays to determine the levels of p‐ EGFR (Tyr1173) and EGFR .
Figure Legend Snippet: Level changes of p‐ EGFR and EGFR in LNC aP and PC 3 cells treated with CFF ‐1 or EGF and co‐treated with CFF ‐1 and EGF . (A and B) LNC aP and PC 3 cells were incubated and treated with increasing doses of CFF ‐1 (0, 2, 5, 10 mg/mL) for 24 h as indicated in figures. And then, cells were harvested for Western blot assays to check the protein levels of p‐ EGFR (Tyr1173), EGFR , and β ‐Actin (loading control). (C and D) LNC aP and PC 3 cells were incubated and treated with 5 mg/mL of CFF ‐1 and then stimulated by different concentrations of EGF (20 μ g/mL, 30 μ g/mL) for 10 min as indicated in figures. Cells were harvested and lysed for Western blot assay to check the protein levels of p‐ EGFR (Tyr1173), EGFR and β ‐Actin (loading control). (E and F) LNC aP and PC 3 cells were pretreated with the indicated concentrations of CFF ‐1 (0, 5, 10 mg/mL) for 24 h and then stimulated by EGF (30 μ g/mL) for 10 min as indicated in figures. Cells were harvested for Western blot assays to determine the levels of p‐ EGFR (Tyr1173) and EGFR .

Techniques Used: Incubation, Western Blot

21) Product Images from "Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer"

Article Title: Deciphering the role of nuclear and cytoplasmic IKKα in skin cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.8792

Characterization of the HaCaT-N-IKKα and HaCaT-C-IKKα cells A-C. Immunofluorescence with a Flag specific antibody showing the expression of the transgene in the nucleus of the HaCaT-N-IKKα cells (B) and in the cytoplasm of the HaCaT-C-IKKα cells (C). D. Representative western blot analyisis showing increased levels of IKKα in different pools of transfected HaCaT clones. Observe the increased MMP-9 and EGFR activation in the HaCaT-C-IKKα cells and the enhanced expression of c-Myc in the HaCaT-N-IKKα cells. E. Graphic representation of the densitometric analysis of western blots correponding to 6 pooled clones of HaCaT-C-IKKα cells, 3 pooled clones of HaCaT-N-IKKα cells and 3 pooled clones of HaCaT-Control cells. Student's t test was used for statistical analysis. (*p
Figure Legend Snippet: Characterization of the HaCaT-N-IKKα and HaCaT-C-IKKα cells A-C. Immunofluorescence with a Flag specific antibody showing the expression of the transgene in the nucleus of the HaCaT-N-IKKα cells (B) and in the cytoplasm of the HaCaT-C-IKKα cells (C). D. Representative western blot analyisis showing increased levels of IKKα in different pools of transfected HaCaT clones. Observe the increased MMP-9 and EGFR activation in the HaCaT-C-IKKα cells and the enhanced expression of c-Myc in the HaCaT-N-IKKα cells. E. Graphic representation of the densitometric analysis of western blots correponding to 6 pooled clones of HaCaT-C-IKKα cells, 3 pooled clones of HaCaT-N-IKKα cells and 3 pooled clones of HaCaT-Control cells. Student's t test was used for statistical analysis. (*p

Techniques Used: Immunofluorescence, Expressing, Western Blot, Transfection, Clone Assay, Activation Assay

Biochemical characterization of Control, C-IKKα and N-IKKα tumors A-F. Representative Western blots analysis of IKKα, P-p65, p65, EGFR, P-EGFR, p100/52, Maspin, c-Myc, E-cadherin and MMP-9 expression in Control, C-IKKα and N-IKKα tumors. Actin and GAPDH were used as loading controls. Western blot of protein extracts from 5 to 8 tumors derived from to 4 to 6 different mice of each genotype were performed. The identification of each tumor and mouse corresponding to every lane is provided in Supp. data G. Bands of the different immunoblots were quantified by Quantity One software and Image Lab software and normalized with respect to Actin or GAPDH expression. P values were determined by Student's t -test and p values
Figure Legend Snippet: Biochemical characterization of Control, C-IKKα and N-IKKα tumors A-F. Representative Western blots analysis of IKKα, P-p65, p65, EGFR, P-EGFR, p100/52, Maspin, c-Myc, E-cadherin and MMP-9 expression in Control, C-IKKα and N-IKKα tumors. Actin and GAPDH were used as loading controls. Western blot of protein extracts from 5 to 8 tumors derived from to 4 to 6 different mice of each genotype were performed. The identification of each tumor and mouse corresponding to every lane is provided in Supp. data G. Bands of the different immunoblots were quantified by Quantity One software and Image Lab software and normalized with respect to Actin or GAPDH expression. P values were determined by Student's t -test and p values

Techniques Used: Western Blot, Expressing, Derivative Assay, Mouse Assay, Software

22) Product Images from "Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB"

Article Title: Docoxahexaenoic Acid Induces Apoptosis of Pancreatic Cancer Cells by Suppressing Activation of STAT3 and NF-κB

Journal: Nutrients

doi: 10.3390/nu10111621

Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.
Figure Legend Snippet: Effect of DHA on the levels of phospho (p)-STAT3, STAT3, p-EGFR, and EGFR, the interaction of EGFR and STAT3, STAT3 DNA-binding activity, and the effect of AG1478 on p-STAT3 and STAT3 levels in PANC-1 cells. Cells were treated with 150 µM DHA for various time periods ( A ) and with 50, 100, and 150 µM DHA for 4 h ( B – D ). The cells were treated with 10 μM tyrophostin AG1478 for 4 h ( E ). ( A ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated with 150 µM DHA for 2, 4, and 6 h. “-” means without DHA treatment and “+” represents with DHA treatment. ( B ) Plot of the protein levels of p-STAT3, STAT3, p-EGFR, and EGFR (and the protein standard actin) in the cells treated for 4 h with the concentrations of DHA indicated. Column “None” corresponds to the extract from untreated cells, column “50”, “100”, and “150” to the extracts from cells treated with 50, 100, and 150 µM DHA, respectively. ( C ) Western blot (WB) of PANC-1 cells treated with 150 µM DHA for 4 h. The total cell lysates were immunoprecipitated (IP) with EGFR antibody and then immunoblotted (by Western blotting, WB) using STAT3 antibody. ( D ) DNA-binding activity of STAT3 in PANC-1 cells treated for 4 h with the indicated concentrations of DHA. The description of the columns is the same as in ( B ). ( E ) Plot of the protein levels of p-STAT3 and STAT3 (and the protein standard actin) in the cells treated with 10 µM AG1478 for 4 h. The column labeled “None” corresponds to the extracts from untreated cells and the column labeled “AG1478” corresponds to the extracts from cells treated with 10 µM AG1478. EGFR = epidermal growth factor receptor (EGFR); STAT3 = signal transducer and activator of transcription factor 3.

Techniques Used: Binding Assay, Activity Assay, Western Blot, Immunoprecipitation, Labeling

23) Product Images from "ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas"

Article Title: ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas

Journal: Oncotarget

doi:

Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.
Figure Legend Snippet: Tumorigenic potential of low passage mucoepidermoid carcinoma cells sorted for ALDH/CD44 A., B. Graphs depicting tumor volume of A. UM-HMC-3A or B. UM-HMC-3B xenograft cells FACS-sorted for ALDH/CD44. Scaffolds were seeded with either 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells and transplanted into the subcutaneous space of SCID mice. Existing tumors were retrieved, re-sorted and 400 ALDH high CD44 high or 4,000 ALDH low CD44 low cells seeded into new scaffolds, and serially passaged in vivo . C. Table depicting the number of tumors grown in the ALDH high CD44 high versus ALDH low CD44 low populations for each passage performed. D. H E staining of tumors generated with FACS-sorted ALDH high CD44 high and ALDH low CD44 low cells. Images were taken at 100X. E. UM-HMC-3A and UM-HMC-3B cells were sorted for ALDH high CD44 high or combined ALDH high CD44 low , ALDH low CD44 high , and ALDH low CD44 low (non-CSC population). NP-40 lysis buffer was used to prepare whole cell lysates that were resolved using PAGE. Membranes were probed using antibodies a 1:1000 dilution against human mTor, p-mTor, Akt, p-Akt, S6K, p-S6K, p-EGFR; 1:2000 dilution of EGFR, and beta-actin.

Techniques Used: FACS, Mouse Assay, In Vivo, Staining, Generated, Lysis, Polyacrylamide Gel Electrophoresis

24) Product Images from "The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries"

Article Title: The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191418

Proposed mechanism of GPER-mediated signaling in porcine coronary artery tension regulation. Activation of GPER by agonist G-1 increases cAMP production and Gβγ release, Gβγ in turn activates tyrosine kinase Src, and then metalloproteinases which cleaves and releases HB-EGF from its precursors ProHB-EGF, and HB-EGF binds and activates EGFR. Active EGFR stimulates its downstream target ERK1/2 and thus leads to contraction.
Figure Legend Snippet: Proposed mechanism of GPER-mediated signaling in porcine coronary artery tension regulation. Activation of GPER by agonist G-1 increases cAMP production and Gβγ release, Gβγ in turn activates tyrosine kinase Src, and then metalloproteinases which cleaves and releases HB-EGF from its precursors ProHB-EGF, and HB-EGF binds and activates EGFR. Active EGFR stimulates its downstream target ERK1/2 and thus leads to contraction.

Techniques Used: Activation Assay

25) Product Images from "CD44 correlates with clinicopathological characteristics and is upregulated by EGFR in breast cancer"

Article Title: CD44 correlates with clinicopathological characteristics and is upregulated by EGFR in breast cancer

Journal: International Journal of Oncology

doi: 10.3892/ijo.2016.3639

Inhibition of EGFR reduced migration and protein abundance of CD44, EMT and CSC-related genes. (A) Transwell assays were utilized to assess the migration. (B) Invasion ability was evaluated by an invasion kit. (C) Western blotting was employed to evaluate the protein levels of EGFR, p-EGFR, p-AKT, p-ERK, CD44, KLF4, Myc, Slug, Snail and Vinculin.
Figure Legend Snippet: Inhibition of EGFR reduced migration and protein abundance of CD44, EMT and CSC-related genes. (A) Transwell assays were utilized to assess the migration. (B) Invasion ability was evaluated by an invasion kit. (C) Western blotting was employed to evaluate the protein levels of EGFR, p-EGFR, p-AKT, p-ERK, CD44, KLF4, Myc, Slug, Snail and Vinculin.

Techniques Used: Inhibition, Migration, Western Blot

Analyses of CD44 in EGFR-negative and EGFR-positive breast tumors. (A) Representative images of CD44 protein abundance in EGFR low and high breast tumors are shown. (B) Semi-quantitative result of CD44 protein abundance in EGFR low and high breast tumors is displayed as the mean ± SE. Oncomine database analysis of the correlation between CD44 mRNA level and EGFR (C), KRT5 (D), KRT17 (E) and FOXA1 (F).
Figure Legend Snippet: Analyses of CD44 in EGFR-negative and EGFR-positive breast tumors. (A) Representative images of CD44 protein abundance in EGFR low and high breast tumors are shown. (B) Semi-quantitative result of CD44 protein abundance in EGFR low and high breast tumors is displayed as the mean ± SE. Oncomine database analysis of the correlation between CD44 mRNA level and EGFR (C), KRT5 (D), KRT17 (E) and FOXA1 (F).

Techniques Used:

26) Product Images from "Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER+ Breast Cancer Cells"

Article Title: Nobiletin Inhibits Angiogenesis by Regulating Src/FAK/STAT3-Mediated Signaling through PXN in ER+ Breast Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18050935

Nobiletin inhibited EGFR activity and Src/FAK/STAT3 signaling. ( A ) Binding of nobiletin (PubChem ID: 72344) to the ATP-binding domain of EGF-R (PDB ID: 2GS2) determined through molecular docking using Autodock vina; ( B ) Western blotting analysis of MCF-7 cells showing that nobiletin inhibited phosphorylated EGFR expression in a concentration-dependent manner; ( C ) MCF-7 cells were pre-treated with recombinant EGF (10 ng/mL) for 10 min and then treated with 200 μM nobiletin followed by isolation of protein and analysis using western blotting; ( D ) Western blotting analysis showing the inhibition of Src/FAK/STAT3 signaling in ER + breast cancer cells after treatment with nobiletin for 24 h; ( E ) Src/FAK/STAT3 signaling inhibition by nobiletin in HUVEC cells by western blotting analysis.
Figure Legend Snippet: Nobiletin inhibited EGFR activity and Src/FAK/STAT3 signaling. ( A ) Binding of nobiletin (PubChem ID: 72344) to the ATP-binding domain of EGF-R (PDB ID: 2GS2) determined through molecular docking using Autodock vina; ( B ) Western blotting analysis of MCF-7 cells showing that nobiletin inhibited phosphorylated EGFR expression in a concentration-dependent manner; ( C ) MCF-7 cells were pre-treated with recombinant EGF (10 ng/mL) for 10 min and then treated with 200 μM nobiletin followed by isolation of protein and analysis using western blotting; ( D ) Western blotting analysis showing the inhibition of Src/FAK/STAT3 signaling in ER + breast cancer cells after treatment with nobiletin for 24 h; ( E ) Src/FAK/STAT3 signaling inhibition by nobiletin in HUVEC cells by western blotting analysis.

Techniques Used: Activity Assay, Binding Assay, Western Blot, Expressing, Concentration Assay, Recombinant, Isolation, Inhibition

27) Product Images from "Hypoxia Changes the Expression of the Epidermal Growth Factor (EGF) System in Human Hearts and Cultured Cardiomyocytes"

Article Title: Hypoxia Changes the Expression of the Epidermal Growth Factor (EGF) System in Human Hearts and Cultured Cardiomyocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040243

mRNA expressions of the EGF receptors EGFR, HER2, HER3, and the JM-b isoform of HER4 in human biopsies. Samples were obtained from a normoxic part of the heart and compared to another sample from the hypoxic part of the same heart. Expressions of mRNA are adjusted by division with the expression of beta-actin. Normoxic tissue expressions are depicted with circular points and hypoxic expression with squares. Each paired sample is connected with a grey dashed line. Data is represented with means and SEM. EGFR shows significant up-regulation from a mean value of 1.0 (normoxic) to a mean value of 2.6 (mean of hypoxic) ( P = 0.03 ). HER2 shows significant down-regulation from 15.0 ((mean of normoxic) to 5.8 ((mean of hypoxic) ( P = 0.0005 ). The differences in HER3 and HER4/JM-b are non-significant ( P = 0.5 and P = 0.4, respectively).
Figure Legend Snippet: mRNA expressions of the EGF receptors EGFR, HER2, HER3, and the JM-b isoform of HER4 in human biopsies. Samples were obtained from a normoxic part of the heart and compared to another sample from the hypoxic part of the same heart. Expressions of mRNA are adjusted by division with the expression of beta-actin. Normoxic tissue expressions are depicted with circular points and hypoxic expression with squares. Each paired sample is connected with a grey dashed line. Data is represented with means and SEM. EGFR shows significant up-regulation from a mean value of 1.0 (normoxic) to a mean value of 2.6 (mean of hypoxic) ( P = 0.03 ). HER2 shows significant down-regulation from 15.0 ((mean of normoxic) to 5.8 ((mean of hypoxic) ( P = 0.0005 ). The differences in HER3 and HER4/JM-b are non-significant ( P = 0.5 and P = 0.4, respectively).

Techniques Used: Expressing

Western Blot showing HL-1 cardiomyocytes exposed to 1% oxygen with or without treatment with trastuzumab. Influence of HB-EGF. Mouse HL-1 cardiomyocytes kept at 1% oxygen were treated either with or without 20 nM trastuzumab for 1 hour followed by treatment with 10 nM HB-EGF for 10 minutes. Whole cell lysates were investigated for phosphorylated and total amounts of the receptors EGFR, HER2, HER3, and HER4. Also examined was the phosphorylation and total amounts of the down-stream signaling molecules MAPK and Akt. Actin was used a loading control. This experiment was repeated twice giving similar results.
Figure Legend Snippet: Western Blot showing HL-1 cardiomyocytes exposed to 1% oxygen with or without treatment with trastuzumab. Influence of HB-EGF. Mouse HL-1 cardiomyocytes kept at 1% oxygen were treated either with or without 20 nM trastuzumab for 1 hour followed by treatment with 10 nM HB-EGF for 10 minutes. Whole cell lysates were investigated for phosphorylated and total amounts of the receptors EGFR, HER2, HER3, and HER4. Also examined was the phosphorylation and total amounts of the down-stream signaling molecules MAPK and Akt. Actin was used a loading control. This experiment was repeated twice giving similar results.

Techniques Used: Western Blot

Regional expression of HB-EGF, EGFR, and HER2 in normal pig hearts. Tissue mRNA expressions are all depicted with circular points. Each paired sample is connected with a grey dashed line. Samples were obtained from the left atrium, the posterior wall of the left ventricle, or the anterior wall of the left ventricle. Data is represented with means and SEM as the ratio between the reference gene GAPDH. HB-EGF, EGFR and HER2 showed no significant regional variation in mRNA expression (all P > 0.05 ).
Figure Legend Snippet: Regional expression of HB-EGF, EGFR, and HER2 in normal pig hearts. Tissue mRNA expressions are all depicted with circular points. Each paired sample is connected with a grey dashed line. Samples were obtained from the left atrium, the posterior wall of the left ventricle, or the anterior wall of the left ventricle. Data is represented with means and SEM as the ratio between the reference gene GAPDH. HB-EGF, EGFR and HER2 showed no significant regional variation in mRNA expression (all P > 0.05 ).

Techniques Used: Expressing

28) Product Images from "Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway"

Article Title: Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-1015-9

Combined treatment of rhein and EGFR inhibitors inhibit tumor growth in xenograft mouse model. a Antitumor efficacy of rhein and erlotinib in the PANC-1 xenograft mouse model. BALB/c mice ( n = 6) were treated with DMSO (Control), 10 mg/kg erlotinib, 60 mg/kg rhein, or the combination. Tumor volumes were recorded every 2 days. b Comparison of the final tumor weights in each group after the 28-day treatment of erlotinib and rhein. Numbers in columns indicate the mean tumor weight in each group. c Photographs of tumors in each group. d Western blot analysis of tumor lysates for phosphorylated EGFR (P-EGFR), phosphorylated STAT3 (P-STAT3), BAX and BCL-2. GAPDH was used as loading control. e Body weight of mice. f No histological abnormalities were observed in kidney, liver, lung and heart in the rhein and erlotinib treated groups. Heart, kidney, lung and liver from the four groups were sectioned at 5 μm, and the slides were stained with hematoxylin and eosin (H E) ( n = 5 in each group). All images were captured using an optical microscope with 200× magnification. All images are representative of three independent experiments. The level of significance is indicated by * P
Figure Legend Snippet: Combined treatment of rhein and EGFR inhibitors inhibit tumor growth in xenograft mouse model. a Antitumor efficacy of rhein and erlotinib in the PANC-1 xenograft mouse model. BALB/c mice ( n = 6) were treated with DMSO (Control), 10 mg/kg erlotinib, 60 mg/kg rhein, or the combination. Tumor volumes were recorded every 2 days. b Comparison of the final tumor weights in each group after the 28-day treatment of erlotinib and rhein. Numbers in columns indicate the mean tumor weight in each group. c Photographs of tumors in each group. d Western blot analysis of tumor lysates for phosphorylated EGFR (P-EGFR), phosphorylated STAT3 (P-STAT3), BAX and BCL-2. GAPDH was used as loading control. e Body weight of mice. f No histological abnormalities were observed in kidney, liver, lung and heart in the rhein and erlotinib treated groups. Heart, kidney, lung and liver from the four groups were sectioned at 5 μm, and the slides were stained with hematoxylin and eosin (H E) ( n = 5 in each group). All images were captured using an optical microscope with 200× magnification. All images are representative of three independent experiments. The level of significance is indicated by * P

Techniques Used: Mouse Assay, Western Blot, Staining, Microscopy

Inhibition of STAT3 by rhein blocks EGFR inhibitors activated STAT3. a PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). Whole-cell protein extracts were analyzed by Western blotting with the indicated antibodies. GAPDH antibody was used as loading control. Figures are representative of three independent experiments. b The PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination. c The pancreatic cell lines BxPC-3 were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). d The BxPC-3 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination
Figure Legend Snippet: Inhibition of STAT3 by rhein blocks EGFR inhibitors activated STAT3. a PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). Whole-cell protein extracts were analyzed by Western blotting with the indicated antibodies. GAPDH antibody was used as loading control. Figures are representative of three independent experiments. b The PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination. c The pancreatic cell lines BxPC-3 were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). d The BxPC-3 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination

Techniques Used: Inhibition, Western Blot

29) Product Images from "Multiple signaling pathways are responsible for prostaglandin E2-induced murine keratinocyte proliferation"

Article Title: Multiple signaling pathways are responsible for prostaglandin E2-induced murine keratinocyte proliferation

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-07-2144

PGE 2 -induces activation of CREB, AP-1 and NF-κB transcription factors in PMKs. PMKs were incubated with vehicle (control, lane 2) or with PGE 2 (10 µM) (lanes 3–7) for 15 min. To demonstrate the effect of phamacological inhibitors on PGE 2 -induced transcription factor-binding, PMKs were treated with PD98059, AG1478, H-89 or wortmannin for 30 min prior to PGE 2 treatment. Nuclear extracts were subjected to electrophoretic mobility shift assay analysis, as described in experimental procedures. A. PGE 2 -induced CREB activation in PMKs. The arrow indicates the specific binding of CREB to its consensus oligonucleotide. B. PGE 2 -induced AP-1 activation in PMKs. The arrow indicates the specific binding of AP-1 to its consensus oligonucleotide. C. PGE 2 -induced NF-κB activation in PMKs. The arrows indicate the specific binding of NF-κB to its consensus oligonucleotide ( upper arrow ) and non-specific (ns) binding ( lower arrow ). D. EGFR, ERK1/2, PKA/CREB and PI3-K/Akt signaling cascades are involved in PGE 2 -stimulated cell proliferation. PMKs were treated with various kinase inhibitors 30 min prior to PGE 2 (10 µM) treatment for 20 h and pulsed with ( 3 H)-thymidine 2 h before harvest. The ( 3 H)-thymidine incorporated by PMKs was measured in triplicate samples and normalized to protein concentration as described in experimental procedures. Representative data from at least 2 independent experiments are presented as the means ± SD. *p
Figure Legend Snippet: PGE 2 -induces activation of CREB, AP-1 and NF-κB transcription factors in PMKs. PMKs were incubated with vehicle (control, lane 2) or with PGE 2 (10 µM) (lanes 3–7) for 15 min. To demonstrate the effect of phamacological inhibitors on PGE 2 -induced transcription factor-binding, PMKs were treated with PD98059, AG1478, H-89 or wortmannin for 30 min prior to PGE 2 treatment. Nuclear extracts were subjected to electrophoretic mobility shift assay analysis, as described in experimental procedures. A. PGE 2 -induced CREB activation in PMKs. The arrow indicates the specific binding of CREB to its consensus oligonucleotide. B. PGE 2 -induced AP-1 activation in PMKs. The arrow indicates the specific binding of AP-1 to its consensus oligonucleotide. C. PGE 2 -induced NF-κB activation in PMKs. The arrows indicate the specific binding of NF-κB to its consensus oligonucleotide ( upper arrow ) and non-specific (ns) binding ( lower arrow ). D. EGFR, ERK1/2, PKA/CREB and PI3-K/Akt signaling cascades are involved in PGE 2 -stimulated cell proliferation. PMKs were treated with various kinase inhibitors 30 min prior to PGE 2 (10 µM) treatment for 20 h and pulsed with ( 3 H)-thymidine 2 h before harvest. The ( 3 H)-thymidine incorporated by PMKs was measured in triplicate samples and normalized to protein concentration as described in experimental procedures. Representative data from at least 2 independent experiments are presented as the means ± SD. *p

Techniques Used: Activation Assay, Incubation, Binding Assay, Electrophoretic Mobility Shift Assay, Protein Concentration

Effect of pharmacological inhibitors on PGE 2 -induced cAMP production and EGFR, c-src, ERK1/2, Akt and PKA/ CREB signaling pathways in PMKs. PMKs were serum starved for 24 h prior to treating with vehicle or PGE 2 (10 µM) for 5 min. Pathway-specific inhibitors were added 30 min before PGE 2 treatment. Western blots of proteins from whole cell lysates were performed with antibodies to the proteins indicated. A. Akt and EGFR inhibitors inhibit PGE 2 -induced Akt activation. Both AG1478 (EGFR inhibitor) and wortmannin (Akt inhibitor) blocked PGE 2 -stimulated phosphorylation of Akt (ser 473 ). B. Effect of MEK, EGFR, and Akt inhibitors on ERK1/2 activation. PD98059 (MEK/ERK inhibitor) completely blocked PGE 2 -stimulated ERK1/2 phosphorylation, while AG1478 and wortmannin were only partially effective. C. Effect of EGFR, c-src, and PKA inhibitors on EGFR activation. AG1478 as expected blocked PGE 2 -stimulated EGFR phosphorylation (tyr 1173 ), while PP2 (c-src inhibitor) and H-89 (PKA inhibitor) had little or no effect. D. EGFR and c-src inhibitors block PGE 2 -induced c-src activation. Both AG1478 and PP2 completely inhibit PGE 2 -stimulated phosphorylation of c-src (tyr 416 ). E. PGE 2 induces cAMP production. Serum-starved PMKs were treated with PGE 2 (0–30 µM) for 30 min with or without a 20 min pretreatment with SQ 22,536 (10 µM), an adenylate cyclase inhibitor. The mean (±SD) levels of cAMP from triplicate samples are expressed as pmol/mg protein. *p
Figure Legend Snippet: Effect of pharmacological inhibitors on PGE 2 -induced cAMP production and EGFR, c-src, ERK1/2, Akt and PKA/ CREB signaling pathways in PMKs. PMKs were serum starved for 24 h prior to treating with vehicle or PGE 2 (10 µM) for 5 min. Pathway-specific inhibitors were added 30 min before PGE 2 treatment. Western blots of proteins from whole cell lysates were performed with antibodies to the proteins indicated. A. Akt and EGFR inhibitors inhibit PGE 2 -induced Akt activation. Both AG1478 (EGFR inhibitor) and wortmannin (Akt inhibitor) blocked PGE 2 -stimulated phosphorylation of Akt (ser 473 ). B. Effect of MEK, EGFR, and Akt inhibitors on ERK1/2 activation. PD98059 (MEK/ERK inhibitor) completely blocked PGE 2 -stimulated ERK1/2 phosphorylation, while AG1478 and wortmannin were only partially effective. C. Effect of EGFR, c-src, and PKA inhibitors on EGFR activation. AG1478 as expected blocked PGE 2 -stimulated EGFR phosphorylation (tyr 1173 ), while PP2 (c-src inhibitor) and H-89 (PKA inhibitor) had little or no effect. D. EGFR and c-src inhibitors block PGE 2 -induced c-src activation. Both AG1478 and PP2 completely inhibit PGE 2 -stimulated phosphorylation of c-src (tyr 416 ). E. PGE 2 induces cAMP production. Serum-starved PMKs were treated with PGE 2 (0–30 µM) for 30 min with or without a 20 min pretreatment with SQ 22,536 (10 µM), an adenylate cyclase inhibitor. The mean (±SD) levels of cAMP from triplicate samples are expressed as pmol/mg protein. *p

Techniques Used: Western Blot, Activation Assay, Blocking Assay

Effect of PGE 2 on keratinocyte proliferation and EGFR, Ras- ERK1/2, and Akt signaling pathways in primary mouse keratinocytes (PMKs). A . PGE 2 increases keratinocyte proliferation in a dose-dependent manner. PMKs from WT mice were treated with PGE 2 (10–30 µM) for 20 h and pulsed with ( 3 H)-thymidine for 2 h before harvest. The ( 3 H)-thymidine incorporated by PMKs was measured and normalized to protein concentration. Data are presented as fold induction of specific activity. At least 2 independent experiments were done, with triplicates for each treatment group; data from a representative experiment are shown as means±SD. *p
Figure Legend Snippet: Effect of PGE 2 on keratinocyte proliferation and EGFR, Ras- ERK1/2, and Akt signaling pathways in primary mouse keratinocytes (PMKs). A . PGE 2 increases keratinocyte proliferation in a dose-dependent manner. PMKs from WT mice were treated with PGE 2 (10–30 µM) for 20 h and pulsed with ( 3 H)-thymidine for 2 h before harvest. The ( 3 H)-thymidine incorporated by PMKs was measured and normalized to protein concentration. Data are presented as fold induction of specific activity. At least 2 independent experiments were done, with triplicates for each treatment group; data from a representative experiment are shown as means±SD. *p

Techniques Used: Mouse Assay, Protein Concentration, Activity Assay

30) Product Images from "S5, a Withanolide Isolated from Physalis Pubescens L., Induces G2/M Cell Cycle Arrest via the EGFR/P38 Pathway in Human Melanoma A375 Cells"

Article Title: S5, a Withanolide Isolated from Physalis Pubescens L., Induces G2/M Cell Cycle Arrest via the EGFR/P38 Pathway in Human Melanoma A375 Cells

Journal: Molecules

doi: 10.3390/molecules23123175

EGFR/P38 signaling pathway was involved in S5-induced cell G2/M arrest. The cells cultured with 40 μM of S5 for 36 h in the absence or presence of 5 μM of SB203580, 40 μg/mL of cetuximab or 5 μM of PD98059. ( A ) The percentage of cells in G2/M phase of the cell cycle was represented using a bar diagram. Data from a representative experiment are shown. n = 3, mean ± SD. * p
Figure Legend Snippet: EGFR/P38 signaling pathway was involved in S5-induced cell G2/M arrest. The cells cultured with 40 μM of S5 for 36 h in the absence or presence of 5 μM of SB203580, 40 μg/mL of cetuximab or 5 μM of PD98059. ( A ) The percentage of cells in G2/M phase of the cell cycle was represented using a bar diagram. Data from a representative experiment are shown. n = 3, mean ± SD. * p

Techniques Used: Cell Culture

ERK, P38 and EGFR are involved in the anti-proliferation of S5 on A375 cells. ( A ) The cells were pretreated with 1.25 μM of SP600125, 5 μM of PD98059, and 5 μM of SB203580 or 40 μg/mL cetuximab for 1 h and then incubated with 40 μM of S5 for 24 h. The death rate of cells was measured using an MTT assay. ( B ) S5 affects the expression of MAPKs and EGFR proteins. The cells were lysed for protein extraction. Samples (25 μg) were subjected to 10% SDS-PAGE and western blotting for the detection of specific proteins. Results presented are the mean from three parallel experiments, n = 3, mean ± SD. * p
Figure Legend Snippet: ERK, P38 and EGFR are involved in the anti-proliferation of S5 on A375 cells. ( A ) The cells were pretreated with 1.25 μM of SP600125, 5 μM of PD98059, and 5 μM of SB203580 or 40 μg/mL cetuximab for 1 h and then incubated with 40 μM of S5 for 24 h. The death rate of cells was measured using an MTT assay. ( B ) S5 affects the expression of MAPKs and EGFR proteins. The cells were lysed for protein extraction. Samples (25 μg) were subjected to 10% SDS-PAGE and western blotting for the detection of specific proteins. Results presented are the mean from three parallel experiments, n = 3, mean ± SD. * p

Techniques Used: Incubation, MTT Assay, Expressing, Protein Extraction, SDS Page, Western Blot

31) Product Images from "JUN-Mediated downregulation of EGFR signaling is associated with resistance to gefitinib in EGFR-mutant NSCLC cell lines"

Article Title: JUN-Mediated downregulation of EGFR signaling is associated with resistance to gefitinib in EGFR-mutant NSCLC cell lines

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-16-0564

JUN expression promotes downregulation of EGFR activity via altered chromatin occupancy (A) Heatmap of differential expression of genes in HCC827 parental and ZDR3 resistant cells (260 genes with log 2 fold change in expression of ± 2.0; p
Figure Legend Snippet: JUN expression promotes downregulation of EGFR activity via altered chromatin occupancy (A) Heatmap of differential expression of genes in HCC827 parental and ZDR3 resistant cells (260 genes with log 2 fold change in expression of ± 2.0; p

Techniques Used: Expressing, Activity Assay

Expression of JUN is inversely correlated with sensitivity to gefitinib (A) HCC827 cells were transduced (MOI=5) with viral particles carrying (GFP) or cDNA for expression of YES1, SRC, JUN, LCK, FYN, or FGR. The levels of each transgene and EGFR were assessed by immunoblot. One representative experiment of three is shown. (B) Cell viability assays were used to determine the differential impact of overexpression of indicated genes on sensitivity to gefitinib. The IC 50 values were calculated using a 4-parameter fit using that statistical package from JMP12 Pro. Each experiment was conducted at three separate time points with at least four technical replicates. Plotted are mean IC 50 values ± one standard deviation from three independent experiments. Probability associated with the Student’s t -test is indicated when values are significant ( p
Figure Legend Snippet: Expression of JUN is inversely correlated with sensitivity to gefitinib (A) HCC827 cells were transduced (MOI=5) with viral particles carrying (GFP) or cDNA for expression of YES1, SRC, JUN, LCK, FYN, or FGR. The levels of each transgene and EGFR were assessed by immunoblot. One representative experiment of three is shown. (B) Cell viability assays were used to determine the differential impact of overexpression of indicated genes on sensitivity to gefitinib. The IC 50 values were calculated using a 4-parameter fit using that statistical package from JMP12 Pro. Each experiment was conducted at three separate time points with at least four technical replicates. Plotted are mean IC 50 values ± one standard deviation from three independent experiments. Probability associated with the Student’s t -test is indicated when values are significant ( p

Techniques Used: Expressing, Over Expression, Standard Deviation

JUN interactomes differ in parental and HCC827-ZDR cells (A) Response to treatment with Gefitinib and JNK inhibitor (AS601245) in HCC827 and HCC827-ZDR3 cell lines (B) Co-IP was used to identify JUN, EGFR and JNK interacting proteins in parental (green) and resistant (red) cell lines. (C) AP-MS was used to identify JUN proteins in parental (green) and resistant (red) cell lines. Proteins that were identified in both cell lines are colored blue. The thickness of the line represents the number of peptides detected. Pie charts indicate proportions of peptides that were identified in each interactome for proteins identified in both cell lines. (D) Co-immunoprecipitation of JUN in HCC827 and HCC827-ZDR3 cells treated with gefitinib (1 µM) or vehicle control (DMSO) for 16 hours. (E) Gene ontology enrichment of JUN interacting proteins identified in both the parental and ZDR3 cell lines. The proteins that were identified in the JUN co-immunoprecipitation experiments were separated into three groups based on the following membership (identified only in parental, ZDR, or both cell lines). Gene ontology enrichment was used to identify biological process that were either unique or shared amongst each group. The abundance of each protein within each group was derived from the number of times each protein was identified (FDR
Figure Legend Snippet: JUN interactomes differ in parental and HCC827-ZDR cells (A) Response to treatment with Gefitinib and JNK inhibitor (AS601245) in HCC827 and HCC827-ZDR3 cell lines (B) Co-IP was used to identify JUN, EGFR and JNK interacting proteins in parental (green) and resistant (red) cell lines. (C) AP-MS was used to identify JUN proteins in parental (green) and resistant (red) cell lines. Proteins that were identified in both cell lines are colored blue. The thickness of the line represents the number of peptides detected. Pie charts indicate proportions of peptides that were identified in each interactome for proteins identified in both cell lines. (D) Co-immunoprecipitation of JUN in HCC827 and HCC827-ZDR3 cells treated with gefitinib (1 µM) or vehicle control (DMSO) for 16 hours. (E) Gene ontology enrichment of JUN interacting proteins identified in both the parental and ZDR3 cell lines. The proteins that were identified in the JUN co-immunoprecipitation experiments were separated into three groups based on the following membership (identified only in parental, ZDR, or both cell lines). Gene ontology enrichment was used to identify biological process that were either unique or shared amongst each group. The abundance of each protein within each group was derived from the number of times each protein was identified (FDR

Techniques Used: Co-Immunoprecipitation Assay, Mass Spectrometry, Immunoprecipitation, Derivative Assay

32) Product Images from "Role of IGF-Binding Protein 3 in the Resistance of EGFR Mutant Lung Cancer Cells to EGFR-Tyrosine Kinase Inhibitors"

Article Title: Role of IGF-Binding Protein 3 in the Resistance of EGFR Mutant Lung Cancer Cells to EGFR-Tyrosine Kinase Inhibitors

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081393

Effects of increased IGFBP3 on the sensitivity to EGFR-TKIs. Resistant cells were infected with Ad/IGFBP-3 at MOIs of 0 to 100 PFU/cell for 48 h and IGFBP-3 expression was determined by Western blotting (A) and ELISA (B). (C) HCC827/GR and HCC827/ER cells were treated with the indicated concentration of EGFR-TKIs for 72 h after infection with 100 MOI of Ad/IGFBP-3. (D) HCC827/GR and HCC827/ER cells were treated with 1 µM EGFR-TKIs and 1 µg/mL rh IGFBP-3 for 72 h. Results are representative of at least three independent experiments, and the error bars represent standard deviation (SD). (E and F) Cells were treated with drugs, rh IGFBP-3 or Ad/IGFBP-3 as in panel C and D. After 24 h, cells were harvested and the modulation of EGFR and IGF1R signalling in the indicated cell lines was detected by Western blotting. (G) Control and IGFBP-3 siRNAs (100 nM) were introduced into HCC827 cells, and IGFBP-3 suppression was confirmed by Western blotting. (H) Cell viability was measured using the MTT assay 72 h later.
Figure Legend Snippet: Effects of increased IGFBP3 on the sensitivity to EGFR-TKIs. Resistant cells were infected with Ad/IGFBP-3 at MOIs of 0 to 100 PFU/cell for 48 h and IGFBP-3 expression was determined by Western blotting (A) and ELISA (B). (C) HCC827/GR and HCC827/ER cells were treated with the indicated concentration of EGFR-TKIs for 72 h after infection with 100 MOI of Ad/IGFBP-3. (D) HCC827/GR and HCC827/ER cells were treated with 1 µM EGFR-TKIs and 1 µg/mL rh IGFBP-3 for 72 h. Results are representative of at least three independent experiments, and the error bars represent standard deviation (SD). (E and F) Cells were treated with drugs, rh IGFBP-3 or Ad/IGFBP-3 as in panel C and D. After 24 h, cells were harvested and the modulation of EGFR and IGF1R signalling in the indicated cell lines was detected by Western blotting. (G) Control and IGFBP-3 siRNAs (100 nM) were introduced into HCC827 cells, and IGFBP-3 suppression was confirmed by Western blotting. (H) Cell viability was measured using the MTT assay 72 h later.

Techniques Used: Infection, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay, Standard Deviation, MTT Assay

33) Product Images from "Novel role of cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways in breast cancer"

Article Title: Novel role of cannabinoid receptor 2 in inhibiting EGF/EGFR and IGF-I/IGF-IR pathways in breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.9408

JWH-015 suppresses ERα- breast cancer growth in vivo by inhibiting EGF/EGFR and IGF-I/IGF-IR signaling pathways A. Tumor volume measurements of orthotopically injected nude mice with SUM159 cells were assessed every week for control and treated groups. B. Tumor weight of vehicle-treated or JWH-015-treated nude mice was determined at the euthanasia day. C. Representative photographs showing tumors dissected from control and treated groups. D. Western blot images of the tumor lysates of the control and treated groups showing the protein expression of phospho-EGFR, phospho-IGF-IR, phospho-STAT3 phospho-ERK and phosphor-AKT (p-EGFR, p-IGF-IR, p-STAT3, p-ERK and p-AKT) and total EGFR, IGF-IR, STAT3, ERK and AKT (EGFR, IGF-IR, STAT3, ERK and AKT).
Figure Legend Snippet: JWH-015 suppresses ERα- breast cancer growth in vivo by inhibiting EGF/EGFR and IGF-I/IGF-IR signaling pathways A. Tumor volume measurements of orthotopically injected nude mice with SUM159 cells were assessed every week for control and treated groups. B. Tumor weight of vehicle-treated or JWH-015-treated nude mice was determined at the euthanasia day. C. Representative photographs showing tumors dissected from control and treated groups. D. Western blot images of the tumor lysates of the control and treated groups showing the protein expression of phospho-EGFR, phospho-IGF-IR, phospho-STAT3 phospho-ERK and phosphor-AKT (p-EGFR, p-IGF-IR, p-STAT3, p-ERK and p-AKT) and total EGFR, IGF-IR, STAT3, ERK and AKT (EGFR, IGF-IR, STAT3, ERK and AKT).

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

Schematic representation of the anti-tumor role of CNR2 activation in breast cancer Schematic representation of the direct anti-tumor role of CNR2 activation showing the possible cross-talk between CNR2 receptor and EGFR and IGF-IR.
Figure Legend Snippet: Schematic representation of the anti-tumor role of CNR2 activation in breast cancer Schematic representation of the direct anti-tumor role of CNR2 activation showing the possible cross-talk between CNR2 receptor and EGFR and IGF-IR.

Techniques Used: Activation Assay

Expression of EGFR, IGF-IR and CNR2 proteins in ERα- and ERα+ breast cancer cells and correlation of CNR2 to breast cancer patient prognosis A. Western blot image shows expression of EGFR, IGF-IR and CNR2 proteins in different ERα- (SCP2, MDA-MB231 and SUM159) and ERα+ (MCF-7 and T47D) breast cancer cell lines. GAPDH was used as a loading control. B. Kaplan Meier blot showing recurrence free survival (RFS) of high/low expressing CNR2 breast cancer patients of ERα- subtype. P value = ~ zero. C. Kaplan Meier blot showing recurrence free survival (RFS) of high/low expressing CNR2 breast cancer patients of ERα+ subtype. P value = 1e −05 .
Figure Legend Snippet: Expression of EGFR, IGF-IR and CNR2 proteins in ERα- and ERα+ breast cancer cells and correlation of CNR2 to breast cancer patient prognosis A. Western blot image shows expression of EGFR, IGF-IR and CNR2 proteins in different ERα- (SCP2, MDA-MB231 and SUM159) and ERα+ (MCF-7 and T47D) breast cancer cell lines. GAPDH was used as a loading control. B. Kaplan Meier blot showing recurrence free survival (RFS) of high/low expressing CNR2 breast cancer patients of ERα- subtype. P value = ~ zero. C. Kaplan Meier blot showing recurrence free survival (RFS) of high/low expressing CNR2 breast cancer patients of ERα+ subtype. P value = 1e −05 .

Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification

34) Product Images from "Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer"

Article Title: Activation of the IGF1R pathway potentially mediates acquired resistance to mutant-selective 3rd-generation EGF receptor tyrosine kinase inhibitors in advanced non-small cell lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.8013

Activation of IGF1R was associated with the resistance to WZ4002 ( A ) Cells were grown to confluence, and then cell lysates were prepared by protein extraction. Phospho-receptor tyrosine kinase array was performed as described in Materials and Methods. ( B ) EGFR and IGF1R-related signal molecules in basal level were assessed using Western blot analysis. ( C ) IGFBP3 mRNA was determined by RT-PCR. ( D ) PC-9/GR/WR cells were treated with MG132 for 6 h. Restored IGFBP3 was determined by Western blot analysis. ( E ) Cells were treated with WZ4002, AG-1024, BI 836845, or a combination of WZ4002 with one of the other 2 drugs for 72 h. Cell viability was measured by MTT assay. ( F ) PC-9/GR/WR cells were treated with drugs as in (E). After 48 h, cells were harvested and subjected to Western blotting using the indicated antibodies. ( G and H ) Lentiviral constructs containing negative control (NT) and IGFR shRNAs were infected into PC-9/GR or PC-9/GR/WR cells, and IGFR silencing was confirmed by Western blot analysis (G). After the selection of puromycin, cells were treated with the indicated doses of WZ4002, and then cell viability was determined by MTT assay (H).
Figure Legend Snippet: Activation of IGF1R was associated with the resistance to WZ4002 ( A ) Cells were grown to confluence, and then cell lysates were prepared by protein extraction. Phospho-receptor tyrosine kinase array was performed as described in Materials and Methods. ( B ) EGFR and IGF1R-related signal molecules in basal level were assessed using Western blot analysis. ( C ) IGFBP3 mRNA was determined by RT-PCR. ( D ) PC-9/GR/WR cells were treated with MG132 for 6 h. Restored IGFBP3 was determined by Western blot analysis. ( E ) Cells were treated with WZ4002, AG-1024, BI 836845, or a combination of WZ4002 with one of the other 2 drugs for 72 h. Cell viability was measured by MTT assay. ( F ) PC-9/GR/WR cells were treated with drugs as in (E). After 48 h, cells were harvested and subjected to Western blotting using the indicated antibodies. ( G and H ) Lentiviral constructs containing negative control (NT) and IGFR shRNAs were infected into PC-9/GR or PC-9/GR/WR cells, and IGFR silencing was confirmed by Western blot analysis (G). After the selection of puromycin, cells were treated with the indicated doses of WZ4002, and then cell viability was determined by MTT assay (H).

Techniques Used: Activation Assay, Protein Extraction, Western Blot, Reverse Transcription Polymerase Chain Reaction, MTT Assay, Construct, Negative Control, Infection, Selection

35) Product Images from "Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway"

Article Title: Rhein sensitizes human pancreatic cancer cells to EGFR inhibitors by inhibiting STAT3 pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-1015-9

Combined treatment of rhein and EGFR inhibitors inhibit tumor growth in xenograft mouse model. a Antitumor efficacy of rhein and erlotinib in the PANC-1 xenograft mouse model. BALB/c mice ( n = 6) were treated with DMSO (Control), 10 mg/kg erlotinib, 60 mg/kg rhein, or the combination. Tumor volumes were recorded every 2 days. b Comparison of the final tumor weights in each group after the 28-day treatment of erlotinib and rhein. Numbers in columns indicate the mean tumor weight in each group. c Photographs of tumors in each group. d Western blot analysis of tumor lysates for phosphorylated EGFR (P-EGFR), phosphorylated STAT3 (P-STAT3), BAX and BCL-2. GAPDH was used as loading control. e Body weight of mice. f No histological abnormalities were observed in kidney, liver, lung and heart in the rhein and erlotinib treated groups. Heart, kidney, lung and liver from the four groups were sectioned at 5 μm, and the slides were stained with hematoxylin and eosin (H E) ( n = 5 in each group). All images were captured using an optical microscope with 200× magnification. All images are representative of three independent experiments. The level of significance is indicated by * P
Figure Legend Snippet: Combined treatment of rhein and EGFR inhibitors inhibit tumor growth in xenograft mouse model. a Antitumor efficacy of rhein and erlotinib in the PANC-1 xenograft mouse model. BALB/c mice ( n = 6) were treated with DMSO (Control), 10 mg/kg erlotinib, 60 mg/kg rhein, or the combination. Tumor volumes were recorded every 2 days. b Comparison of the final tumor weights in each group after the 28-day treatment of erlotinib and rhein. Numbers in columns indicate the mean tumor weight in each group. c Photographs of tumors in each group. d Western blot analysis of tumor lysates for phosphorylated EGFR (P-EGFR), phosphorylated STAT3 (P-STAT3), BAX and BCL-2. GAPDH was used as loading control. e Body weight of mice. f No histological abnormalities were observed in kidney, liver, lung and heart in the rhein and erlotinib treated groups. Heart, kidney, lung and liver from the four groups were sectioned at 5 μm, and the slides were stained with hematoxylin and eosin (H E) ( n = 5 in each group). All images were captured using an optical microscope with 200× magnification. All images are representative of three independent experiments. The level of significance is indicated by * P

Techniques Used: Mouse Assay, Western Blot, Staining, Microscopy

Inhibition of STAT3 by rhein blocks EGFR inhibitors activated STAT3. a PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). Whole-cell protein extracts were analyzed by Western blotting with the indicated antibodies. GAPDH antibody was used as loading control. Figures are representative of three independent experiments. b The PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination. c The pancreatic cell lines BxPC-3 were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). d The BxPC-3 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination
Figure Legend Snippet: Inhibition of STAT3 by rhein blocks EGFR inhibitors activated STAT3. a PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). Whole-cell protein extracts were analyzed by Western blotting with the indicated antibodies. GAPDH antibody was used as loading control. Figures are representative of three independent experiments. b The PANC-1 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination. c The pancreatic cell lines BxPC-3 were treated with DMSO (Control), 60 μM rhein, 5 μM erlotinib or the combination of both inhibitors (rhein + erlotinib). d The BxPC-3 cells were treated with DMSO (Control), 60 μM rhein, 2.5 μM afatinib or the combination

Techniques Used: Inhibition, Western Blot

36) Product Images from "Grandinin down-regulates phosphorylation of epidermal growth factor receptor"

Article Title: Grandinin down-regulates phosphorylation of epidermal growth factor receptor

Journal: International Journal of Clinical and Experimental Pathology

doi:

Quantitative RT-PCR analysis of the mRNA expression levels of p-EGFR and p-AKT in SBC3, MS-1, A549, and LK-2 cells. One microliter of RNA for each condition was transcribed into cDNA using random primers in a Reverse Transcription II system (Promega,
Figure Legend Snippet: Quantitative RT-PCR analysis of the mRNA expression levels of p-EGFR and p-AKT in SBC3, MS-1, A549, and LK-2 cells. One microliter of RNA for each condition was transcribed into cDNA using random primers in a Reverse Transcription II system (Promega,

Techniques Used: Quantitative RT-PCR, Expressing, Mass Spectrometry

Grandinin decreases expression of p-EGFR and p-AKT. SBC3, MS-1, A549, and LK-2 cells were treated with either vehicle control (DMSO) or grandinin (16 μM) for 48 h. Whole-cell extracts were prepared and western blot analyses were performed to analyze
Figure Legend Snippet: Grandinin decreases expression of p-EGFR and p-AKT. SBC3, MS-1, A549, and LK-2 cells were treated with either vehicle control (DMSO) or grandinin (16 μM) for 48 h. Whole-cell extracts were prepared and western blot analyses were performed to analyze

Techniques Used: Expressing, Mass Spectrometry, Western Blot

37) Product Images from "AUY922 Effectively Overcomes MET- and AXL-Mediated Resistance to EGFR-TKI in Lung Cancer Cells"

Article Title: AUY922 Effectively Overcomes MET- and AXL-Mediated Resistance to EGFR-TKI in Lung Cancer Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0119832

Suppression of MET and AXL by AUY922 in resistant cell lines. A , Cells were treated with the indicated doses of AUY922 for 72 hours in medium containing 1% FBS. Attached cells were stained with trypan blue solution (top). Cell viability based on cell counting is also shown (bottom). Bars represent the mean ±SD of three wells. B , Cells treated with AUY922, similar to panel A. After 48 hours, cells were harvested and EGFR-related signaling molecules were evaluated using western blotting. C , LK2 cells were treated with vector-containing wild-type EGFR, del E746-E750, MET, or AXL and the indicated doses of AUY922 for 12 hours.
Figure Legend Snippet: Suppression of MET and AXL by AUY922 in resistant cell lines. A , Cells were treated with the indicated doses of AUY922 for 72 hours in medium containing 1% FBS. Attached cells were stained with trypan blue solution (top). Cell viability based on cell counting is also shown (bottom). Bars represent the mean ±SD of three wells. B , Cells treated with AUY922, similar to panel A. After 48 hours, cells were harvested and EGFR-related signaling molecules were evaluated using western blotting. C , LK2 cells were treated with vector-containing wild-type EGFR, del E746-E750, MET, or AXL and the indicated doses of AUY922 for 12 hours.

Techniques Used: Staining, Cell Counting, Western Blot, Plasmid Preparation

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Article Snippet: .. The primary antibodies, EGFR (1005) (Santa Cruz), p-EGFR (Tyr 1173) (Santa Cruz), Sox2 (R & D systems), Nestin (BD), GFAP (ImmunO), NICD (Cell signaling), Hes1 (Millipore), Hey1 (abcam), PEDF (Upstate), and β-actin (Santa Cruz) were incubated overnight at 4°C. .. Immunoreactive bands were visualized using peroxidase-labeled affinity purified secondary antibodies (KPL) and the detection reagent Amersham ECL prime western blotting detection reagent (GE Healthcare).

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    Santa Cruz Biotechnology p egfr
    Inhibition of <t>EGFR</t> pathway blocked <t>ERK/AKT/cyclin</t> D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2
    P Egfr, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CBD inhibits <t>EGF/EGFR</t> signaling. (A) SUM159 cells were treated with vehicle or CBD 6 μM in the presence or absence of EGF and subjected to NF‐kB luciferase reporter assay. (B) SUM159 cells were treated with CBD 6 μM,
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    Inhibition of EGFR pathway blocked ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2

    Journal: Photochemistry and photobiology

    Article Title: Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human Keratinocytes

    doi: 10.1111/j.1751-1097.2008.00531.x

    Figure Lengend Snippet: Inhibition of EGFR pathway blocked ERK/AKT/cyclin D1 pathways and cell cycle progression induced by UVB exposure. (A) Cells were incubated with or without AG1478 (1 µ M ), an EGFR kinase inhibitor, and then exposed to UVB radiation (10 mJ cm −2

    Article Snippet: Antibodies used were as follows: cyclin D1 (Cell Signal), p-AKT (phospho-AKT-ser473; Cell Signal); p-ERK (phospho-ERK1/2; Santa Cruz); p-EGFR (phosphor-EGFR-tyrosine 1173; Santa Cruz); AKT (Santa Cruz), ERK (Santa Cruz), EGFR (Neo-marker) and β-actin (Santa Cruz).

    Techniques: Inhibition, Incubation

    Cells were exposed to UVB as in . Cells were harvested at 1.5, 3 or 6 h following UVB radiation for Western blot analysis using specific antibodies against cyclin D1, p-AKT, AKT, p-ERK, ERK, p-EGFR, EGFR and beta-actin (an equal loading control)

    Journal: Photochemistry and photobiology

    Article Title: Requirement for Metalloproteinase-dependent ERK and AKT Activation in UVB-induced G1-S Cell Cycle Progression of Human Keratinocytes

    doi: 10.1111/j.1751-1097.2008.00531.x

    Figure Lengend Snippet: Cells were exposed to UVB as in . Cells were harvested at 1.5, 3 or 6 h following UVB radiation for Western blot analysis using specific antibodies against cyclin D1, p-AKT, AKT, p-ERK, ERK, p-EGFR, EGFR and beta-actin (an equal loading control)

    Article Snippet: Antibodies used were as follows: cyclin D1 (Cell Signal), p-AKT (phospho-AKT-ser473; Cell Signal); p-ERK (phospho-ERK1/2; Santa Cruz); p-EGFR (phosphor-EGFR-tyrosine 1173; Santa Cruz); AKT (Santa Cruz), ERK (Santa Cruz), EGFR (Neo-marker) and β-actin (Santa Cruz).

    Techniques: Western Blot

    Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P

    Journal: Oncogene

    Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    doi: 10.1038/onc.2009.234

    Figure Lengend Snippet: Overexpression of a NLS tagged EGFR in cetuximab-resistant cells confers resistance to cetuximab in vivo Male athymic nude mice were injected subcutaneously with 1×10 6 cetuximab-sensitive parental cells (HP) or CMV-EGFR-NLS/Myc clone cells (Vector only, Clone 4, Clone 5 and Clone 10) into the dorsal flank. Once tumors reached a volume 120-180mm 3 mice were treated with 0.1 mg IgG or cetuximab twice weekly. Tumor diameters were measured serially with calipers and tumor volumes were calculated. Points, mean tumor volume of eight mice per group; bars, SD. T -test was used to compare tumor volumes between cetuximab treated and control IgG mice. *, P

    Article Snippet: The antibodies used in this study were as follows: EGFR, p-EGFR (Tyr1173), CyclinD1, B-Myb, HRP-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Over Expression, In Vivo, Mouse Assay, Injection, Plasmid Preparation

    Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.

    Journal: Oncogene

    Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    doi: 10.1038/onc.2009.234

    Figure Lengend Snippet: Potential mechanism for resistance to cetuximab A ) Cetuximab-sensitive cells depend on classical EGFR membrane signaling. B ) Tumor cells that acquire resistance to cetuximab gain nEGFR as a second compartment of proliferation. C ) Cetuximab can abrogate signals from plasma membrane EGFR but not nEGFR; nEGFR continues to send proliferative signals by modulation of Cyclin D1, B-myb, Aurora kinase K and regulation of PCNA. D ) The SFK inhibitor dasatinib inhibits nuclear translocation of the EGFR from the plasma membrane leading to increased EGFR on the plasma membrane and restoring sensitivity to cetuximab.

    Article Snippet: The antibodies used in this study were as follows: EGFR, p-EGFR (Tyr1173), CyclinD1, B-Myb, HRP-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay

    Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P

    Journal: Oncogene

    Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    doi: 10.1038/onc.2009.234

    Figure Lengend Snippet: Src family kinases mediate ligand-induced EGFR translocation to the nucleus A ) Dasatinib inhibits HER family ligands signaling in parental cells (HP). HP cells were untreated, treated for 24 hours with 50 nM of dasatinib alone, or followed by 200 ng/ml of indicated ligand for 1 hour prior to harvesting. Nuclear protein was collected and fractionated by SDS-PAGE followed by immunoblotting for EGFR. histone H3 was used as loading control. B ) Dasatinib inhibits nuclear expression of EGFR in cetuximab-resistant cell lines. Parental cells (HP) and cetuximab-resistant cell lines (HC1, HC4, HC8) were treated with 50 nM of dasatinib for 24 hours. After cells were harvested, cytoplasmic and nuclear protein was fractionated by SDS-PAGE followed by immunoblotting for EGFR. α-tubulin and histone H3 were used as loading controls and purity controls of each cellular fraction. Expression of nEGFR after dasatinib treatment in cetuximab-resistant clones was quantitated using ImageJ software and normalized against the amounts of untreated cells. C ) Dasatinib treatment lead to increased membrane-bound EGFR in cetuximab-resistant cells by flow cytometry analysis. Parental cells (HP) and cetuximab-resistant cells (HC1, HC4 and HC8) were treated with DMSO or 50 nM of dasatinib for 24 hours and membrane expression is represented relative to untreated controls. Mean surface expression of EGFR is represented +/- SEM (n=3). Flow cytometric plots of representative experiments are presented. Shaded histograms represent dasatinib treatment. Controls (dotted line) represent cells labeled with FITC-conjugated normal mouse IgG *, P

    Article Snippet: The antibodies used in this study were as follows: EGFR, p-EGFR (Tyr1173), CyclinD1, B-Myb, HRP-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Translocation Assay, SDS Page, Expressing, Clone Assay, Software, Flow Cytometry, Cytometry, Labeling

    EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P

    Journal: Oncogene

    Article Title: Nuclear EGFR Contributes to Acquired Resistance to Cetuximab

    doi: 10.1038/onc.2009.234

    Figure Lengend Snippet: EGFR tagged with nuclear localization sequence confers resistance to cetuximab in vitro A ) A schematic representation of the CMV-EGFR-NLS/Myc construct is shown. EGFR-NLS/Myc was driven by the CMV promoter. The cetuximab-sensitive NSCLC line NCI-H226 was infected with indicated constructs. Represented is three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). Cytoplasmic and nuclear protein from each clone was collected and immunoprecipitated with an anti-myc antibody, fractionated on SDS-PAGE and immunoblotted with the indicated antibodies. α-tubulin and histone H3 were used as loading and purity control for cytosolic and nuclear fractions, respectively. Immunofluorescence of nEGFR staining in CMV-EGFR-NLS/Myc clones. EGFR (green), DNA (blue), stained by PI. V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. 400× magnification. cEGFR; cytoplasmic EGFR, nEGFR; nuclear EGFR. B ) CMV-EGFR-NLS/Myc expressed in NCI-H226 leads to increased cyclin D1 and B-myb expression. Nuclear protein from EGFR-NLS/myc clones was collected and fractionated by SDS-PAGE followed by immunoblotting for the indicated proteins. histone H3 was used as a loading control. Expression of cyclin D1 and B-myb in CMV-EGFR-NLS/Myc clones (C 4 , C 5 and C 10 ) were quantitated using ImageJ software and normalized against the amounts of those proteins in vector control (V 0 ). V 0 ; vector clone, C 4 , C 5 and C 10 ; CMV-EGFR-NLS/Myc clones. C ) Growth response to cetuximab of three individual clones and vector control (V 0 ; vector only, C 4 , C 5 and C 10 ). CMV-EGFR-NLS/Myc-tag clones (C 4 , C 5 and C 10 ) were treated with 100 nM of cetuximab and growth was measured using the growth proliferation assay and plotted as growth relative to untreated control. Data points are represented as mean +/- SEM. (n=3). *, P

    Article Snippet: The antibodies used in this study were as follows: EGFR, p-EGFR (Tyr1173), CyclinD1, B-Myb, HRP-conjugated goat-anti-rabbit IgG and goat-anti-mouse IgG were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

    Techniques: Sequencing, In Vitro, Construct, Infection, Clone Assay, Plasmid Preparation, Immunoprecipitation, SDS Page, Immunofluorescence, Staining, Expressing, Software, Proliferation Assay

    EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P

    Journal: Oncotarget

    Article Title: WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression

    doi:

    Figure Lengend Snippet: EGFR transactivation is involved in WISP-1-induced VEGF-A expression and contributing to angiogenesis (A–D) SCC4 cells were pre-treated with an EGFR inhibitor (AG1478; 1 μM) for 30 min or transfected with EGFR siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (E–H) SCC4 cells were treated by an ERK inhibitor (U0126; 1 μM) for 30 min or transfected with ERK siRNAs for 24 h, followed by WISP-1 (20 ng/mL) stimulation for 24 h. The assay procedures were performed as described in Figure 3A–3D . (I) SCC4 cells were incubated with WISP-1 (20 ng/mL) for the indicated times and EGFR and ERK phosphorylation was determined by western blot. (J–K) SCC4 cells were incubated with the integrin αvβ3 antibody, FAKi, PP2, or AG1478 for 30 min, followed by stimulation with WISP-1 (20 ng/mL) for 60 min, and EGFR (J) and ERK (K) phosphorylation was determined by western blot. Data are expressed as the mean ± SEM * P

    Article Snippet: Materials Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-FAK, FAK, p-c-Src, c-Src, p-EGFR, EGFR, p-ERK, ERK, HIF1-α, β-actin, CD31, and WISP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGF-A antibody was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Transfection, Incubation, Western Blot

    WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P

    Journal: Oncotarget

    Article Title: WISP-1, a novel angiogenic regulator of the CCN family, promotes oral squamous cell carcinoma angiogenesis through VEGF-A expression

    doi:

    Figure Lengend Snippet: WISP-1 knockdown in OSCC decreases VEGF-A expression and angiogenesis-related tumor growth in vivo (A) SCC4 cells stably expressing shRNA constructs or control shRNA were seeded as monolayers and counted daily. Cells (10 3 ) were plated in 6 well plates and grown for 2 days. Cells were trypsinized, and cell numbers was counted. (B–C) WISP-1 and VEGF-A mRNA and protein expression in SCC4 cells stably expressed a control shRNA or a WISP-1 shRNA was examined by western blot, qPCR, and ELISA. (D–E) EPCs were incubated with CM collected from control-shRNA and WISP-1-shRNA transfected SCC4 cells for 24 h and cell migration or tube formation were examined. (F) PBS, VEGF-A, control shRNA/SCC4 CM, and WISP-1 shRNA/SCC4 CM mixed in Matrigel were placed on chick chorioallantoic membranes. CAMs in each group were photographed on developmental day 12. (G) Mice were subcutaneously injected with Matrigel mixed with PBS, control shRNA/SCC4 CM or WISP-1 shRNA/SCC4 CM for seven days. Plugs excised from the mice were photographed and stained with CD31. (H) Control shRNA and WISP-1 shRNA SCC4 cells were mixed with Matrigel and injected into the flank of the mice for 28 days. Tumor growth was monitored using the IVIS Imaging System. Tumor growth was quantified by fluorescent imaging from week 0–6. (I) Tumors were paraffin embedded, and sections were immunostained using the WISP-1, VEGF-A, and CD31 antibodies. (E = epithelial, T = tumor, S = stroma). (J) Diagrammatic model for the role of WISP-1 in OSCC. (1) WISP-1 induces VEGF-A expression and secretion in OSCC cells through the integrin αvβ3/FAK/c-Src pathway, which transactivates the EGFR/ERK/HIF1-α signal pathway. (2) The WISP-1-induced secretion of VEGF-A subsequently recruiting EPCs to OSCC tumor microenvironment and promoting neoangiogenesis. Data represent the mean ± SEM * P

    Article Snippet: Materials Anti-mouse and anti-rabbit IgG-conjugated horseradish peroxidase, rabbit polyclonal antibodies specific for p-FAK, FAK, p-c-Src, c-Src, p-EGFR, EGFR, p-ERK, ERK, HIF1-α, β-actin, CD31, and WISP-1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); VEGF-A antibody was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, In Vivo, Stable Transfection, shRNA, Construct, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Migration, Mouse Assay, Injection, Staining, Imaging

    CBD inhibits EGF/EGFR signaling. (A) SUM159 cells were treated with vehicle or CBD 6 μM in the presence or absence of EGF and subjected to NF‐kB luciferase reporter assay. (B) SUM159 cells were treated with CBD 6 μM,

    Journal: Molecular Oncology

    Article Title: Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway; novel anti-tumor mechanisms of Cannabidiol in breast cancer

    doi: 10.1016/j.molonc.2014.12.010

    Figure Lengend Snippet: CBD inhibits EGF/EGFR signaling. (A) SUM159 cells were treated with vehicle or CBD 6 μM in the presence or absence of EGF and subjected to NF‐kB luciferase reporter assay. (B) SUM159 cells were treated with CBD 6 μM,

    Article Snippet: The following reagents and antibodies used in this study were purchased from different sources: Cannabidiol (Sigma Aldrich); human/murine EGF (Peprotech); GAPDH, AKT, p‐EGFR/EGFR, Arginase‐I, CD31, and p‐ERK/ERK (Santa Cruz); and F4/80 (Abd Serotec) and Ki67 from (NeoMarkers); and p‐AKT from (Cell Signaling).

    Techniques: Luciferase, Reporter Assay