Structured Review

Cell Signaling Technology Inc total egfr
Mutations at the site of drug binding cause resistance in drug sensitive <t>EGFR</t> or <t>HER2</t> exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.
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1) Product Images from "Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR and HER2 inhibitors"

Article Title: Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR and HER2 inhibitors

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-16-3404

Mutations at the site of drug binding cause resistance in drug sensitive EGFR or HER2 exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.
Figure Legend Snippet: Mutations at the site of drug binding cause resistance in drug sensitive EGFR or HER2 exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

Techniques Used: Binding Assay, Mutagenesis, Expressing, Construct

EGFR and ERBB2 exon 20 insertions and sensitivity to covalent quinazoline based EGFR inhibitors. A. Ba/F3 cells expressing wild type EGFR or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing different EGFR exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. Ba/F3 cells expressing wild type HER2 or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing different HER2 exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.
Figure Legend Snippet: EGFR and ERBB2 exon 20 insertions and sensitivity to covalent quinazoline based EGFR inhibitors. A. Ba/F3 cells expressing wild type EGFR or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing different EGFR exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. Ba/F3 cells expressing wild type HER2 or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing different HER2 exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

Techniques Used: Expressing

2) Product Images from "Enhanced cohesion promotes chromosome stability and limits acquired drug resistance in non small cell lung cancer"

Article Title: Enhanced cohesion promotes chromosome stability and limits acquired drug resistance in non small cell lung cancer

Journal: bioRxiv

doi: 10.1101/2020.07.10.197350

Suppression of CIN influences mechanisms of drug tolerance. A) Quantification of anaphase cells exhibiting lagging chromosomes in clonal populations of drug tolerant cells with (Wapl KD) or without (Mock) Wapl depletion. B C) Gefitinib-tolerant PC9 cell clones derived with or without Wapl depletion labelled with centromere enumeration FISH probes for chromosomes 6, 7, 8, and 10 and quantified for intratumor numerical heterogeneity. D) Western blot showing Wapl levels and EGFR pathway status in four each of mock and Wapl-depleted drug tolerant clones. E) qPCR analysis of mRNA levels indicative of EMT pathway activation (slug, vimentin) and MET expression in mock and Wapl-depleted drug tolerant clones.
Figure Legend Snippet: Suppression of CIN influences mechanisms of drug tolerance. A) Quantification of anaphase cells exhibiting lagging chromosomes in clonal populations of drug tolerant cells with (Wapl KD) or without (Mock) Wapl depletion. B C) Gefitinib-tolerant PC9 cell clones derived with or without Wapl depletion labelled with centromere enumeration FISH probes for chromosomes 6, 7, 8, and 10 and quantified for intratumor numerical heterogeneity. D) Western blot showing Wapl levels and EGFR pathway status in four each of mock and Wapl-depleted drug tolerant clones. E) qPCR analysis of mRNA levels indicative of EMT pathway activation (slug, vimentin) and MET expression in mock and Wapl-depleted drug tolerant clones.

Techniques Used: Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Western Blot, Real-time Polymerase Chain Reaction, Activation Assay, Expressing

3) Product Images from "Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells"

Article Title: Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbalip.2016.04.006

DCA activation of ERK1/2 requires EGFR but does not result in phosphorylation of the receptor at Tyr1068
Figure Legend Snippet: DCA activation of ERK1/2 requires EGFR but does not result in phosphorylation of the receptor at Tyr1068

Techniques Used: Activation Assay

4) Product Images from "Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis"

Article Title: Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis

Journal: Mucosal immunology

doi: 10.1038/mi.2017.10

ITLN1 contributes to HDM-induced IL-25, IL-33 and TSLP expression and the activation of EGFR and ERK pathway in human bronchial epithelial cells. ( a – d ) The kinetics of ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells after exposure to HDM were determined by quantitative PCR. n = 4 wells per group. Data are mean ± SEM. **, P
Figure Legend Snippet: ITLN1 contributes to HDM-induced IL-25, IL-33 and TSLP expression and the activation of EGFR and ERK pathway in human bronchial epithelial cells. ( a – d ) The kinetics of ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells after exposure to HDM were determined by quantitative PCR. n = 4 wells per group. Data are mean ± SEM. **, P

Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction

5) Product Images from "Fibrinogen inhibits neurite outgrowth via ?3 integrin-mediated phosphorylation of the EGF receptor"

Article Title: Fibrinogen inhibits neurite outgrowth via ?3 integrin-mediated phosphorylation of the EGF receptor

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.0704045104

Proposed model of fibrinogen-mediated inhibition of neurite outgrowth. Traumatic injury or other neurodegenerative conditions associated with compromised BBB or vascular damage allow the leakage of fibrinogen in the CNS. Fibrinogen binding to β3 integrin on neurons induces coclustering of β3 integrin with EGFR, leading to EGFR phosphorylation and subsequent inhibition of neurite outgrowth.
Figure Legend Snippet: Proposed model of fibrinogen-mediated inhibition of neurite outgrowth. Traumatic injury or other neurodegenerative conditions associated with compromised BBB or vascular damage allow the leakage of fibrinogen in the CNS. Fibrinogen binding to β3 integrin on neurons induces coclustering of β3 integrin with EGFR, leading to EGFR phosphorylation and subsequent inhibition of neurite outgrowth.

Techniques Used: Inhibition, Binding Assay

Fibrinogen induces transactivation of EGFR via β3 integrin to inhibit neurite outgrowth. ( A and B ) Treatment with the EGFR phosphorylation inhibitor PD168393 (50 nM) on 1.5 mg/ml fibrinogen treatment results in a statistically significant increase in the percentage of mouse CGNs bearing neurites ( A ; P
Figure Legend Snippet: Fibrinogen induces transactivation of EGFR via β3 integrin to inhibit neurite outgrowth. ( A and B ) Treatment with the EGFR phosphorylation inhibitor PD168393 (50 nM) on 1.5 mg/ml fibrinogen treatment results in a statistically significant increase in the percentage of mouse CGNs bearing neurites ( A ; P

Techniques Used:

6) Product Images from "JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors"

Article Title: JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors

Journal: Science signaling

doi: 10.1126/scisignal.aac8460

JAK2 inhibition increases surface EGFR expression through SOCS5 ( A ) Detection of cell surface–bound EGFR on PC-9R cells using Alexa Fluor–EGF at 4°C after a 1-hour treatment with AZD1480 or control. Scale bars, 50 μm. ( B . ( C ) Detection of JAK2-EGFR and SOCS5-EGFR interactions by Duolink staining in PC-9R cells treated with AZD1480 or control for 1 hour. Scale bars, 50 μm. ( D . ( E ) PC-9R cells expressing JAK2 shRNA (JAK2sh) or vector control (Csh) constructs analyzed for SOCS5 and EGFR interactions by Duolink staining. Scale bars, 50 μm. Cell lysates were analyzed for JAK2 and tubulin. ( F ) Western blot for SOCS5 and tubulin in lysates from H1975 cells expressing scrambled control or SOCS5 shRNA (SOCS5sh). Control or SOCS5sh cells were treated with control, AZD1480 (1 μM), or erlotinib (0.2 μM) for 1 hour and analyzed for pEGFR, EGFR, and tubulin by Western blot. Representative blots are shown ( n = 3). ( G ) Tumor volumes in mice bearing H1975-SOCS5Sh xenografts and treated with vehicle or TKI (25 mg/kg per day) for 9 days. Data are means ± SEM ( n = 5 to 7 mice per group). ** P
Figure Legend Snippet: JAK2 inhibition increases surface EGFR expression through SOCS5 ( A ) Detection of cell surface–bound EGFR on PC-9R cells using Alexa Fluor–EGF at 4°C after a 1-hour treatment with AZD1480 or control. Scale bars, 50 μm. ( B . ( C ) Detection of JAK2-EGFR and SOCS5-EGFR interactions by Duolink staining in PC-9R cells treated with AZD1480 or control for 1 hour. Scale bars, 50 μm. ( D . ( E ) PC-9R cells expressing JAK2 shRNA (JAK2sh) or vector control (Csh) constructs analyzed for SOCS5 and EGFR interactions by Duolink staining. Scale bars, 50 μm. Cell lysates were analyzed for JAK2 and tubulin. ( F ) Western blot for SOCS5 and tubulin in lysates from H1975 cells expressing scrambled control or SOCS5 shRNA (SOCS5sh). Control or SOCS5sh cells were treated with control, AZD1480 (1 μM), or erlotinib (0.2 μM) for 1 hour and analyzed for pEGFR, EGFR, and tubulin by Western blot. Representative blots are shown ( n = 3). ( G ) Tumor volumes in mice bearing H1975-SOCS5Sh xenografts and treated with vehicle or TKI (25 mg/kg per day) for 9 days. Data are means ± SEM ( n = 5 to 7 mice per group). ** P

Techniques Used: Inhibition, Expressing, Staining, shRNA, Plasmid Preparation, Cell Surface Hydrophobicity, Construct, Western Blot, Mouse Assay

JAK inhibition or depletion enhances EGFR-ERK signaling ( A . ( B ) Staining for pSTAT3, pEGFR, EGFR, and pERK in representative tumor sections from H1650 xenografts treated with vehicle control (C) or AZD1480 (Ji) (30 mg/kg, twice daily for 3 weeks). Scale bars, 100 μm. ( C .
Figure Legend Snippet: JAK inhibition or depletion enhances EGFR-ERK signaling ( A . ( B ) Staining for pSTAT3, pEGFR, EGFR, and pERK in representative tumor sections from H1650 xenografts treated with vehicle control (C) or AZD1480 (Ji) (30 mg/kg, twice daily for 3 weeks). Scale bars, 100 μm. ( C .

Techniques Used: Inhibition, Staining

Enhancement of EGFR-ERK signaling by JAK inhibition is mediated through heterodimerization between wild-type/mutant EGFR, which is abrogated by TKI ( A ) Serum-starved H1975 cells were pretreated with AZD1480 or control (C) for 1 hour, and EGF ligand was added for the indicated times. Surface proteins were biotinylated, precipitated with avidin resin beads, and analyzed by Western blot for surface EGFR (sEGFR) and c-MET (sMET). ( B ) Duolink staining (left) for wild-type (WT) EGFR (Myc) and mutant L858R EGFR (MUT) interaction in H1975 cells expressing Myc-tagged WT EGFR protein and treated with AZD1480 or control for 1 hour. Scale bars, 50 μm. Western blot (right) in lysates from H1975 parental cells (control) and cells expressing Myc-tagged WT EGFR were analyzed for EGFR, Myc-tagged protein, and tubulin. ( C ) Top, experimental schematic in which H1975 cells expressing both the WT (blue sphere) and EGFR-L858R/T790M gatekeeper mutant (red sphere) proteins are depicted as a single cell expressing variable amounts of each. H1975 cells were treated with either TKI (0.2 μM) or the T790M-specific inhibitor WZ4002 (WZ; 25 nM) for 30 days, and selected populations are depicted by their relative expression of WT and mutant EGFR per cell. Extracts from WZ4002- and TKI-selected cells treated with either control or AZD1480 were then analyzed by Western blot as indicated (note that EGFR detects both WT and mutant). The growth inhibitory effects of AZD1480 (JAKi) in combination with erlotinib (TKI) are shown below as representative CIs. ( D , respectively.
Figure Legend Snippet: Enhancement of EGFR-ERK signaling by JAK inhibition is mediated through heterodimerization between wild-type/mutant EGFR, which is abrogated by TKI ( A ) Serum-starved H1975 cells were pretreated with AZD1480 or control (C) for 1 hour, and EGF ligand was added for the indicated times. Surface proteins were biotinylated, precipitated with avidin resin beads, and analyzed by Western blot for surface EGFR (sEGFR) and c-MET (sMET). ( B ) Duolink staining (left) for wild-type (WT) EGFR (Myc) and mutant L858R EGFR (MUT) interaction in H1975 cells expressing Myc-tagged WT EGFR protein and treated with AZD1480 or control for 1 hour. Scale bars, 50 μm. Western blot (right) in lysates from H1975 parental cells (control) and cells expressing Myc-tagged WT EGFR were analyzed for EGFR, Myc-tagged protein, and tubulin. ( C ) Top, experimental schematic in which H1975 cells expressing both the WT (blue sphere) and EGFR-L858R/T790M gatekeeper mutant (red sphere) proteins are depicted as a single cell expressing variable amounts of each. H1975 cells were treated with either TKI (0.2 μM) or the T790M-specific inhibitor WZ4002 (WZ; 25 nM) for 30 days, and selected populations are depicted by their relative expression of WT and mutant EGFR per cell. Extracts from WZ4002- and TKI-selected cells treated with either control or AZD1480 were then analyzed by Western blot as indicated (note that EGFR detects both WT and mutant). The growth inhibitory effects of AZD1480 (JAKi) in combination with erlotinib (TKI) are shown below as representative CIs. ( D , respectively.

Techniques Used: Inhibition, Mutagenesis, Avidin-Biotin Assay, Western Blot, Staining, Expressing

Working model: JAK inhibition enhances TKI-sensitive EGFR signaling and growth inhibition in NSCLC Schematic depicting TKI-resistant NSCLC cells expressing both WT and gatekeeper mutant EGFR proteins, which form homodimers or heterodimers. At steady state, JAK2 promotes SOCS5-dependent EGFR degradation. TKI-resistant, homodimeric mutant EGFR is the principle driver of the RAS–MEK (mitogen-activated protein kinase kinase)–ERK signaling cascade (in bold). Inhibition or reduction of JAK2 uncouples SOCS5 from EGFR, effectively increasing TKI-sensitive, wild-type EGFR homodimers/heterodimers and signaling (in bold). Although the role of EGFR WT:MUT heterodimer signaling has not been clearly defined, we hypothesize that it may regulate TKI sensitivity. This working model may explain the synergistic actions observed between the EGFR-targeted TKI and JAKi on NSCLC tumor growth.
Figure Legend Snippet: Working model: JAK inhibition enhances TKI-sensitive EGFR signaling and growth inhibition in NSCLC Schematic depicting TKI-resistant NSCLC cells expressing both WT and gatekeeper mutant EGFR proteins, which form homodimers or heterodimers. At steady state, JAK2 promotes SOCS5-dependent EGFR degradation. TKI-resistant, homodimeric mutant EGFR is the principle driver of the RAS–MEK (mitogen-activated protein kinase kinase)–ERK signaling cascade (in bold). Inhibition or reduction of JAK2 uncouples SOCS5 from EGFR, effectively increasing TKI-sensitive, wild-type EGFR homodimers/heterodimers and signaling (in bold). Although the role of EGFR WT:MUT heterodimer signaling has not been clearly defined, we hypothesize that it may regulate TKI sensitivity. This working model may explain the synergistic actions observed between the EGFR-targeted TKI and JAKi on NSCLC tumor growth.

Techniques Used: Inhibition, Expressing, Mutagenesis

7) Product Images from "Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models"

Article Title: Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0820-5

Working model of intra-pathway feedbacks and BRAF/MEK growth-inhibitory synergism. a In BRAF-wt/KRAS-mut contexts, selective BRAF inhibition induces BRAF-CRAF dimerization, which hyperactivates the MAPK pathway, thus resulting in relative resistance to treatment. In BRAF-wt/KRAS-wt contexts, paradoxical MAPK activation may be sustained by the RAS-dependent upstream signaling of RTKs (in particular EGFR family members). b Upon allosteric MEK inhibition, the MAPK pathway downstream of a mutant KRAS is efficiently shut down; however, MEK inhibition-induced removal of ERK-mediated feedback RTK inhibition may result in incomplete MAPK pathway inhibition or pathway reactivation, again resulting in relative resistance to the drug. c Combined BRAF/MEK inhibition results in efficient pathway blockade and a synergistic effect on cell growth inhibition, particularly downstream of a mutant KRAS; however, as highlighted also in panel b, in KRAS-wt contexts removal of ERK-mediated feedback RTK inhibition may result in RTK-dependent pathway (re)activation, thus resulting in only partial blockade of downstream signaling. d Thus, in KRAS-wt contexts, triple RTK (EGFR family in the specific case discussed here)/BRAF/MEK inhibition is hypothesized to completely prevent paradoxical MAPK activation; functional growth-inhibitory synergism will then vary according to the degree of intrinsic sensitivity/resistance to RTK inhibition
Figure Legend Snippet: Working model of intra-pathway feedbacks and BRAF/MEK growth-inhibitory synergism. a In BRAF-wt/KRAS-mut contexts, selective BRAF inhibition induces BRAF-CRAF dimerization, which hyperactivates the MAPK pathway, thus resulting in relative resistance to treatment. In BRAF-wt/KRAS-wt contexts, paradoxical MAPK activation may be sustained by the RAS-dependent upstream signaling of RTKs (in particular EGFR family members). b Upon allosteric MEK inhibition, the MAPK pathway downstream of a mutant KRAS is efficiently shut down; however, MEK inhibition-induced removal of ERK-mediated feedback RTK inhibition may result in incomplete MAPK pathway inhibition or pathway reactivation, again resulting in relative resistance to the drug. c Combined BRAF/MEK inhibition results in efficient pathway blockade and a synergistic effect on cell growth inhibition, particularly downstream of a mutant KRAS; however, as highlighted also in panel b, in KRAS-wt contexts removal of ERK-mediated feedback RTK inhibition may result in RTK-dependent pathway (re)activation, thus resulting in only partial blockade of downstream signaling. d Thus, in KRAS-wt contexts, triple RTK (EGFR family in the specific case discussed here)/BRAF/MEK inhibition is hypothesized to completely prevent paradoxical MAPK activation; functional growth-inhibitory synergism will then vary according to the degree of intrinsic sensitivity/resistance to RTK inhibition

Techniques Used: Inhibition, Activation Assay, Mutagenesis, Functional Assay

Selective BRAF inhibition induces EGFR family-dependent MAPK hyperactivation in Calu-3 cells. a and b The NSCLC cell line Calu-3 ( HER2 -amplified, KRAS -wt) was treated with increasing concentrations of dabrafenib (0.01–10 μM) alone a or in combination with trametinib (ratio 1:1000; b ) for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the proteins indicated. Western blot with Hsp70 specific antibody is shown as protein loading and blotting control. c Calu-3 cells were treated with increasing concentrations of dabrafenib (0.01–10 μM) and trametinib (0.01 nM–10 nM) alone or in combination for 72 h. Cell viability was assessed by Crystal violet assay and pharmacologic interactions were evaluated using the Calcusyn software. The results represent the average ± SD of three independent experiments. d Calu-3 and HCC827 ( EGFR -mut) cells were treated with increasing concentrations of dabrafenib (0.1–10 μM) and lapatinib (0.1–10 μM) for 4 h. The proteins were subjected to Western Blotting and analyzed for the indicated antibodies. e MiaPaCa2, A549 and A427 cells were treated with dabrafenib (10 μM) and lapatinib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated
Figure Legend Snippet: Selective BRAF inhibition induces EGFR family-dependent MAPK hyperactivation in Calu-3 cells. a and b The NSCLC cell line Calu-3 ( HER2 -amplified, KRAS -wt) was treated with increasing concentrations of dabrafenib (0.01–10 μM) alone a or in combination with trametinib (ratio 1:1000; b ) for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the proteins indicated. Western blot with Hsp70 specific antibody is shown as protein loading and blotting control. c Calu-3 cells were treated with increasing concentrations of dabrafenib (0.01–10 μM) and trametinib (0.01 nM–10 nM) alone or in combination for 72 h. Cell viability was assessed by Crystal violet assay and pharmacologic interactions were evaluated using the Calcusyn software. The results represent the average ± SD of three independent experiments. d Calu-3 and HCC827 ( EGFR -mut) cells were treated with increasing concentrations of dabrafenib (0.1–10 μM) and lapatinib (0.1–10 μM) for 4 h. The proteins were subjected to Western Blotting and analyzed for the indicated antibodies. e MiaPaCa2, A549 and A427 cells were treated with dabrafenib (10 μM) and lapatinib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated

Techniques Used: Inhibition, Amplification, Western Blot, Crystal Violet Assay, Software

8) Product Images from "Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR mutant lung cancer"

Article Title: Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR mutant lung cancer

Journal: bioRxiv

doi: 10.1101/2020.10.26.356352

RBM10 modulates the apoptotic response to osimertinib in EGFR mutant lung adenocarcinoma. A-D, H3255 and PC-9 ( EGFR mutant and RBM10 wt) cells expressing shRBM10 or shScramble control were treated with the third-generation EGFR inhibitor osimertinib (500 nM) or DMSO for 48 to 72 hours. Western blot analysis of the indicated proteins from cellular protein extracts was normalized to actin; quantification of cleaved PARP was determined by signal densitometry ( A, B ). Apoptotic response was assessed using a Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. ( C, D ). E-F, RBM10-deficient A014 ( EGFR mutant and RBM10 Q255 *) cells with genetic reconstitution of WT RBM10 were treated with osimertinib (500 nM) for 48 hours. Western blotting of indicated lysates was normalized to actin ( E ). Caspase-3/7 activity was measured using a Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. ( F ). Data represent 3 independent experiments. O.E.:Overexpression. *; p
Figure Legend Snippet: RBM10 modulates the apoptotic response to osimertinib in EGFR mutant lung adenocarcinoma. A-D, H3255 and PC-9 ( EGFR mutant and RBM10 wt) cells expressing shRBM10 or shScramble control were treated with the third-generation EGFR inhibitor osimertinib (500 nM) or DMSO for 48 to 72 hours. Western blot analysis of the indicated proteins from cellular protein extracts was normalized to actin; quantification of cleaved PARP was determined by signal densitometry ( A, B ). Apoptotic response was assessed using a Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. ( C, D ). E-F, RBM10-deficient A014 ( EGFR mutant and RBM10 Q255 *) cells with genetic reconstitution of WT RBM10 were treated with osimertinib (500 nM) for 48 hours. Western blotting of indicated lysates was normalized to actin ( E ). Caspase-3/7 activity was measured using a Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. ( F ). Data represent 3 independent experiments. O.E.:Overexpression. *; p

Techniques Used: Mutagenesis, Expressing, Western Blot, Caspase-Glo Assay, Activity Assay, Over Expression

RBM10 deficiency decreases the Bcl-xS/Bcl-xL ratio to limit the apoptotic response to EGFR TKI therapy. A, RBM10 regulates Bcl-x mRNA splicing into Bcl-xS (pro-apoptotic) and Bcl-xL (anti-apoptotic) isoforms. B-C, Quantitative RT-PCR analysis of Bcl-xS-to-Bcl-xL ratio (mRNA levels) in H3255 ( B ) and PC-9 ( C ) cells expressing shRBM10 or shScr control. Data are shown as mean ± SEM of the fold change after normalization to housekeeping gene (GAPDH). D-E, Conventional PCR analysis using validated primers to detect both Bcl-xL and Bcl-xS isoforms in H3255 and PC-9 cells expressing either shScr control, shRBM10 with or without genetic rescue of Bcl-xS . F-G, H3255 and PC-9 ( EGFR L858R, EGFR del19 respectively; RBM10 WT) cells treated with osimertinib for 48 and 72 hours, which express either shRBM10 or shScr control paired with or without genetic rescue of Bcl-xS. Cell lysates were harvested, and the indicated proteins were determined by western blot analysis. H-I, Caspase-3/7 activity was measured using Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. J-K, Quantitative RT-PCR ( J ) and conventional RT-PCR analysis ( K ) of the Bcl-xS-to-Bcl-xL ratio (mRNA levels) following genetic reconstitution of RBM10 or Bcl-xS in RBM10-deficient A014 cells. L, A014 cells (RBM10 deficient) overexpressing Bcl-xS or reconstituted with RBM10 24h before the osimertinib (500 nM) treatment or DMSO control for 48 hours. Cell lysates were harvested, and the indicated proteins were determined by western blot analysis. M, Activity of Caspase-3/7 was measured using Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. Data represent 3 independent experiments. O.E.: Overexpression. BP; Base Pairs. *; p
Figure Legend Snippet: RBM10 deficiency decreases the Bcl-xS/Bcl-xL ratio to limit the apoptotic response to EGFR TKI therapy. A, RBM10 regulates Bcl-x mRNA splicing into Bcl-xS (pro-apoptotic) and Bcl-xL (anti-apoptotic) isoforms. B-C, Quantitative RT-PCR analysis of Bcl-xS-to-Bcl-xL ratio (mRNA levels) in H3255 ( B ) and PC-9 ( C ) cells expressing shRBM10 or shScr control. Data are shown as mean ± SEM of the fold change after normalization to housekeeping gene (GAPDH). D-E, Conventional PCR analysis using validated primers to detect both Bcl-xL and Bcl-xS isoforms in H3255 and PC-9 cells expressing either shScr control, shRBM10 with or without genetic rescue of Bcl-xS . F-G, H3255 and PC-9 ( EGFR L858R, EGFR del19 respectively; RBM10 WT) cells treated with osimertinib for 48 and 72 hours, which express either shRBM10 or shScr control paired with or without genetic rescue of Bcl-xS. Cell lysates were harvested, and the indicated proteins were determined by western blot analysis. H-I, Caspase-3/7 activity was measured using Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. J-K, Quantitative RT-PCR ( J ) and conventional RT-PCR analysis ( K ) of the Bcl-xS-to-Bcl-xL ratio (mRNA levels) following genetic reconstitution of RBM10 or Bcl-xS in RBM10-deficient A014 cells. L, A014 cells (RBM10 deficient) overexpressing Bcl-xS or reconstituted with RBM10 24h before the osimertinib (500 nM) treatment or DMSO control for 48 hours. Cell lysates were harvested, and the indicated proteins were determined by western blot analysis. M, Activity of Caspase-3/7 was measured using Caspase-Glo 3/7 assay. Each bar represents the mean ± SEM of the fold change after normalization to DMSO control. Data represent 3 independent experiments. O.E.: Overexpression. BP; Base Pairs. *; p

Techniques Used: Quantitative RT-PCR, Expressing, Polymerase Chain Reaction, Western Blot, Activity Assay, Caspase-Glo Assay, Reverse Transcription Polymerase Chain Reaction, Over Expression

RBM10 deficiency limits the therapeutic efficacy of EGFR TKIs. A-D, Waterfall plots representing immunodeficient mice bearing H3255 ( A, C ) or PC-9 ( B, D ) tumor xenografts expressing either shScramble (shScr) control or sh RBM10 ; mice were treated with 2 mg/kg (H3255) or 5mg/kg (PC-9) osimertinib once daily over a duration of 14 days (n=10 tumors per treatment cohort). Percent changes in tumor volume compared to baseline volume (Day 0) for individual tumor xenografts are shown ( A, B ). Objective tumor response was graded using RECIST response criteria, comparing H3255 and PC-9 tumors expressing either shScr control or shRBM10 treated with osimertinib ( C, D ). E-F, H3255 and PC-9 tumor xenograft explants demonstrating the effect of RBM10 knockdown on PARP cleavage in mice treated with osimertinib or vehicle for 14 days. Tumors were harvested 4 hours after indicated treatments, and subsequent analyses of the indicated proteins was performed by western blot. G-I, PC-9 cells expressing either shScr or shRBM10 in a validated orthotopic lung tumor model were treated with 5 mg/kg osimertinib once daily for 60 days. Representative bioluminescent images ( G ) and mean relative photon flux ( H ) are shown. Progression-free survival (PFS) comparing PC-9 shScr control and PC-9 shRBM10 mice is shown ( I ) (p-value = 0.0002, Wilcoxon test). *; p
Figure Legend Snippet: RBM10 deficiency limits the therapeutic efficacy of EGFR TKIs. A-D, Waterfall plots representing immunodeficient mice bearing H3255 ( A, C ) or PC-9 ( B, D ) tumor xenografts expressing either shScramble (shScr) control or sh RBM10 ; mice were treated with 2 mg/kg (H3255) or 5mg/kg (PC-9) osimertinib once daily over a duration of 14 days (n=10 tumors per treatment cohort). Percent changes in tumor volume compared to baseline volume (Day 0) for individual tumor xenografts are shown ( A, B ). Objective tumor response was graded using RECIST response criteria, comparing H3255 and PC-9 tumors expressing either shScr control or shRBM10 treated with osimertinib ( C, D ). E-F, H3255 and PC-9 tumor xenograft explants demonstrating the effect of RBM10 knockdown on PARP cleavage in mice treated with osimertinib or vehicle for 14 days. Tumors were harvested 4 hours after indicated treatments, and subsequent analyses of the indicated proteins was performed by western blot. G-I, PC-9 cells expressing either shScr or shRBM10 in a validated orthotopic lung tumor model were treated with 5 mg/kg osimertinib once daily for 60 days. Representative bioluminescent images ( G ) and mean relative photon flux ( H ) are shown. Progression-free survival (PFS) comparing PC-9 shScr control and PC-9 shRBM10 mice is shown ( I ) (p-value = 0.0002, Wilcoxon test). *; p

Techniques Used: Mouse Assay, Expressing, Western Blot, Significance Assay

Resistance caused by RBM10 deficiency in EGFR mutant lung cancer can be overcome with Bcl-xL and EGFR inhibitor combination therapy. A-F, RBM10-defιcient A014 ( EGFR L858R; RBM10 Q255 *) and H1975 ( EGFR L8585R/T790M; RBM10 G840fs*7 ) cells were treated with navitoclax (ABT-263) 500nM alone or in combination with indicated osimertinib concentrations. Crystal violet viability assays ( A, B ) and apoptosis was measured with PARP cleavage and Caspase 3/7 activity ( C-F ). Each bar represents the mean ± SEM of the fold change after normalization to DMSO control ( E, F ). G-H , Western blot analysis of Bcl-xL knockdown with siRNA in combination with osimertinib 500nM in A014 and H1975 cells. I-J, Mice bearing H1975 subcutaneous xenografts were treated with vehicle, navitoclax (50mg/kg), osimertinib (5mg/kg), or combination (navitoclax and osimertinib) therapy for 14 days (n=10 tumors each arm). Percent changes in tumor volume compared to baseline for individual xenografts are shown. Objective tumor response for indicated treatment groups in ( J ). K, H1975 xenograft tumor explants were treated with vehicle, navitoclax, osimertinib, or combination (navitoclax and osimertinib, 50mg/kg and 5mg/kg respectively) therapy for 14 days. Tumors were harvested 4 hours after treatment and the indicated protein levels were determined by Western blot analysis. L, Proposed model of RBM10 regulating EGFR TKI-induced apoptosis through differential Bcl-x splicing. Data represent 3 independent experiments in data panels. *; p
Figure Legend Snippet: Resistance caused by RBM10 deficiency in EGFR mutant lung cancer can be overcome with Bcl-xL and EGFR inhibitor combination therapy. A-F, RBM10-defιcient A014 ( EGFR L858R; RBM10 Q255 *) and H1975 ( EGFR L8585R/T790M; RBM10 G840fs*7 ) cells were treated with navitoclax (ABT-263) 500nM alone or in combination with indicated osimertinib concentrations. Crystal violet viability assays ( A, B ) and apoptosis was measured with PARP cleavage and Caspase 3/7 activity ( C-F ). Each bar represents the mean ± SEM of the fold change after normalization to DMSO control ( E, F ). G-H , Western blot analysis of Bcl-xL knockdown with siRNA in combination with osimertinib 500nM in A014 and H1975 cells. I-J, Mice bearing H1975 subcutaneous xenografts were treated with vehicle, navitoclax (50mg/kg), osimertinib (5mg/kg), or combination (navitoclax and osimertinib) therapy for 14 days (n=10 tumors each arm). Percent changes in tumor volume compared to baseline for individual xenografts are shown. Objective tumor response for indicated treatment groups in ( J ). K, H1975 xenograft tumor explants were treated with vehicle, navitoclax, osimertinib, or combination (navitoclax and osimertinib, 50mg/kg and 5mg/kg respectively) therapy for 14 days. Tumors were harvested 4 hours after treatment and the indicated protein levels were determined by Western blot analysis. L, Proposed model of RBM10 regulating EGFR TKI-induced apoptosis through differential Bcl-x splicing. Data represent 3 independent experiments in data panels. *; p

Techniques Used: Mutagenesis, Activity Assay, Western Blot, Mouse Assay

9) Product Images from "The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L"

Article Title: The histone H3K9 demethylase KDM3A promotes anoikis by transcriptionally activating pro-apoptotic genes BNIP3 and BNIP3L

Journal: eLife

doi: 10.7554/eLife.16844

Inhibition of FAK, EGFR, or MEK in MCF10A cells increases KDM3A expression. (A–C) qRT-PCR analysis monitoring KDM3A expression in MCF10A cells treated for 48 hr with 0, 1, 5 or 10 µM FAK inhibitor ( A ), gefitinib ( B ), or U0126 ( C ). Error bars indicate SD. **p
Figure Legend Snippet: Inhibition of FAK, EGFR, or MEK in MCF10A cells increases KDM3A expression. (A–C) qRT-PCR analysis monitoring KDM3A expression in MCF10A cells treated for 48 hr with 0, 1, 5 or 10 µM FAK inhibitor ( A ), gefitinib ( B ), or U0126 ( C ). Error bars indicate SD. **p

Techniques Used: Inhibition, Expressing, Quantitative RT-PCR

10) Product Images from "Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer"

Article Title: Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer

Journal: BMC Cancer

doi: 10.1186/1471-2407-13-518

EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).
Figure Legend Snippet: EGF receptor is autophosphorylated in Caco-2 and activates downstream signalling pathways. Caco-2 cells were stimulated with 20 ng/mL EGF for the time periods indicated. Western blotting for (a) phosphorylated EGFR or total EGFR and (b) antibodies recognising signalling enzymes is shown. Cell lysate of EGF-treated A431 cells was used as positive control. α-tubulin is shown as a loading control. Densitometry was performed using Phoretix 1D analysis software against α-tubulin (for ERK, data for p42 and p44 are shown).

Techniques Used: Western Blot, Positive Control, Software

11) Product Images from "Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation"

Article Title: Celecoxib increases EGF signaling in colon tumor associated fibroblasts, modulating EGFR expression and degradation

Journal: Oncotarget

doi:

Celecoxib increases EGFR mRNA and protein expression a ) Real time PCR for EGFR. Colon TAFs were treated with Celecoxib (Cel, 10μM) for 48 hours; EGF (50ng/ml) was added as indicated during the last 16h of incubation. EGFR mRNA levels were normalized against the RP2 housekeeping gene. The mean values of three independent tests are shown. b ) A western blot for EGFR under the same condition reported for Real Time PCR. c ) The relative mean intensity of bands from six independent western blots, on three different TAFs primary cell cultures, was calculated by densitometry and plotted. d ) Flow cytometric analysis of surface and total EGFR. TAFs were treated as described above. The peaks, representing EGFR expression under Celecoxib (Cel), EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. e ) Surface and total EGFR increase induced by Celecoxib, calculated as Cel+EGF / EGF ratio of three independent flow cytometric analyses as reported in panel d. f ) Binding and internalization of biotin-EGF in colon TAFs primed or not with Celecoxib and treated with biotin-EGF (50ng/ml) for 45 or 30min respectively. The test was run in six replicates and repeated three times.
Figure Legend Snippet: Celecoxib increases EGFR mRNA and protein expression a ) Real time PCR for EGFR. Colon TAFs were treated with Celecoxib (Cel, 10μM) for 48 hours; EGF (50ng/ml) was added as indicated during the last 16h of incubation. EGFR mRNA levels were normalized against the RP2 housekeeping gene. The mean values of three independent tests are shown. b ) A western blot for EGFR under the same condition reported for Real Time PCR. c ) The relative mean intensity of bands from six independent western blots, on three different TAFs primary cell cultures, was calculated by densitometry and plotted. d ) Flow cytometric analysis of surface and total EGFR. TAFs were treated as described above. The peaks, representing EGFR expression under Celecoxib (Cel), EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. e ) Surface and total EGFR increase induced by Celecoxib, calculated as Cel+EGF / EGF ratio of three independent flow cytometric analyses as reported in panel d. f ) Binding and internalization of biotin-EGF in colon TAFs primed or not with Celecoxib and treated with biotin-EGF (50ng/ml) for 45 or 30min respectively. The test was run in six replicates and repeated three times.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Flow Cytometry, Binding Assay

Celecoxib retards Cathepsin-D maturation in late endosomes, causing EGFR and Rab7 accumulation a ) Cytoplasmic vesicles from TAFs treated as shown were fractioned by centrifugation (see methods) obtaining samples enriched in lysosomes (Lys: EEA1 low; Rab7, LAMP1 high; Cathepsin-D very high), late endosomes (LE: EEA1 low; Cathepsin middle; Rab7, LAMP1 high) and early endosomes (EE: EEA1 high; Rab7, LAMP1, Cathepsin-D low). Post nuclear supernatants (Pns) from untreated controls were used as loading controls. b - e ) Relative quantification of EGFR, Rab7, pro Cathepsin-D and active Cathepsin-D bands in Lys, LE and EE-enriched fractions from replicates of the test shown in panel a. f) EGFR, Rab7 and pro/active Cathepsin-D gain in the Lys, LE and EE-enriched fractions of Celecoxib-treated samples, calculated from data shown in graphs b-e as ratios against the relative controls (both untreated or EGF-treated).
Figure Legend Snippet: Celecoxib retards Cathepsin-D maturation in late endosomes, causing EGFR and Rab7 accumulation a ) Cytoplasmic vesicles from TAFs treated as shown were fractioned by centrifugation (see methods) obtaining samples enriched in lysosomes (Lys: EEA1 low; Rab7, LAMP1 high; Cathepsin-D very high), late endosomes (LE: EEA1 low; Cathepsin middle; Rab7, LAMP1 high) and early endosomes (EE: EEA1 high; Rab7, LAMP1, Cathepsin-D low). Post nuclear supernatants (Pns) from untreated controls were used as loading controls. b - e ) Relative quantification of EGFR, Rab7, pro Cathepsin-D and active Cathepsin-D bands in Lys, LE and EE-enriched fractions from replicates of the test shown in panel a. f) EGFR, Rab7 and pro/active Cathepsin-D gain in the Lys, LE and EE-enriched fractions of Celecoxib-treated samples, calculated from data shown in graphs b-e as ratios against the relative controls (both untreated or EGF-treated).

Techniques Used: Centrifugation

Celecoxib slows down EGFR degradation a ) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b ) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c ) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d ) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e ) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.
Figure Legend Snippet: Celecoxib slows down EGFR degradation a ) Western blot analysis of the kinetic of EGFR degradation. Colon TAFs pretreated with Celecoxib were challenged with EGF for the indicated times. The arrow indicates the band used for EGFR degradation quantification. The test was repeated twice. b ) The early endosome marker 1 (EEA1) levels were not influenced by Celecoxib pretreatment. c ) A representative image (90min EGF) of double immunofluorescence analyses: EGFR (red), EEA1 (green). Celecoxib-pretreated colon TAFs were challenged with EGF for 30, 90, 180min or 16h. Fluorescent images were acquired, with fixed expositions (EEA1-488 f1/8; EGFR-594 f1/3; DAPI f1/100), by a Leica DM-LB2 microscope equipped with I3 and M2 filters and a HCX PL Fluotar 40x non immersion optic. A 20μm scale is shown. d ) Analysis of Mander's overlay coefficients for EGFR and EEA1 on the double immunofluorescence. Six random 40x fields per condition -containing at least 12 TAFs- were analyzed (see methods). The test was repeated twice. e ) Flow cytometric analysis for EGFR expression in TAFs pretreated or not with Celecoxib and then challenged for 90 or 180min with EGF. The peaks, representing EGFR expression under EGF, or Celecoxib plus EGF (Cel+EGF) treatments (white peaks), were compared to EGFR levels detected in untreated controls (grey peaks). The MFI ratio (white/grey) was calculated and reported on each panel. The test was repeated twice.

Techniques Used: Western Blot, Marker, Immunofluorescence, Microscopy, Flow Cytometry, Expressing

Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.
Figure Legend Snippet: Celecoxib affects protein turnover mimicking the inhibitors of endo-lysosome acidification a ) Western blot for EGFR and p62/SQSTM1 of colon TAFs pretreated with Celecoxib and triggered for 3 or 16h with EGF. b ) Western blot analysis of the effects of proteasome-lysosome inhibitors. 48h treatment with MG132 (proteasome inhibitor, 2μM), Bafilomycin-A1 (Baf, V-ATPase inhibitor, 25nM), NH 4 Cl (lysosomal pH -buffering molecule, 10mM) were compared to Celecoxib (10μM) as modulators of EGFR, p62, HSP70 and IkBα. c ) Relative quantification of EGFR and p62 levels from replicate tests as shown in panel b; p values were calculated as compared to untreated controls. d ) Western blot comparison of the effects of Celecoxib and lysosome acidification inhibitors. The effects of Celecoxib (10μM), Bafilomycin-A1 (2.5nM) and NH 4 Cl (2.5mM) pretreatment were tested in the absence/presence of EGF (3h) on several target proteins: EGFR, p62, IkBα, EEA1, LAMP1, Rab7, pro Cathepsin-D and Cathepsin-D. Loading controls: beta-actin (1) normalizes EEA1, p62 and Cathepsin-D; beta-actin (2) normalizes EGFR, IkBα, LAMP1 and Rab7. e ) Relative quantification of EGFR, p62, Rab7, pro and active Cathepsin-D levels from replicate tests as shown in panel d; p values defined in Materials and Methods were calculated as compared to untreated controls.

Techniques Used: Western Blot

12) Product Images from "Celastrol Induces Apoptosis in Gefitinib-Resistant Non-Small Cell Lung Cancer Cells via Caspases-Dependent Pathways and Hsp90 Client Protein Degradation"

Article Title: Celastrol Induces Apoptosis in Gefitinib-Resistant Non-Small Cell Lung Cancer Cells via Caspases-Dependent Pathways and Hsp90 Client Protein Degradation

Journal: Molecules

doi: 10.3390/molecules19033508

Celastrol significantly induced the degradation of Hsp90 client proteins: EGFR and AKT in both H1650 and H1975.
Figure Legend Snippet: Celastrol significantly induced the degradation of Hsp90 client proteins: EGFR and AKT in both H1650 and H1975.

Techniques Used:

13) Product Images from "Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2"

Article Title: Human breast cancer cells harboring a gatekeeper T798M mutation in HER2 overexpress EGFR ligands and are sensitive to dual inhibition of EGFR and HER2

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-13-1038

HER2 T798M cells express higher levels of EGFR ligands and HER2-containing heterodimers A . RT qPCR analysis for ErbB receptor ligand expression was performed using RNA template from BT474 GFP and BT474 T798M cells, normalized to expression in GFP cells for each ligand. B . Lysates prepared from BT474 GFP (± 40 ng/ml TGFα) and BT474 T798M were immunoprecipitated with a HER2 C-terminal antibody followed by immunoblot analyses with the indicated antibodies. C . Lysates prepared from MCF10A WT (± 40 ng/ml TGFα) and MCF10A T798M were analyzed as in B. D . BT474 T798M cells were treated with 20 μg/ml cetuximab and 1 μM lapatinib, either alone or in combination for 5 days. MTT assays were performed at the end of treatment. E . Cells were treated with 20 μg/ml cetuximab and/or 1 μM lapatinib for 3 h. Lysates were prepared and subjected to immunoblot analyses with the indicated antibodies. F . For 3D Matrigel growth assays, cells were treated with 20 μg/ml trastuzumab, 20 μg/ml cetuximab or the combination. Cells were plated in duplicates and images captured on day 14. Fresh medium and inhibitors were replenished every 3 days. G . Average acinar area was quantified with the ImageJ software. Each bar represents the mean acinar area ± S.E.M ( n =2). H . Cells were treated with 20 μg/ml cetuximab and/or and 20 μg/ml trastuzumab and lysates analyzed as in E. I . BT474 T798M xenografts were established in female athymic mice as indicated in Methods. Once tumors reached at least ≥200 mm 3 in volume, mice were treated with trastuzumab (30 mg/kg i.p. twice per week), cetuximab (1 mg i.p. twice per week) or both antibodies. Each data point represents the mean tumor volume ± SEM. *, p
Figure Legend Snippet: HER2 T798M cells express higher levels of EGFR ligands and HER2-containing heterodimers A . RT qPCR analysis for ErbB receptor ligand expression was performed using RNA template from BT474 GFP and BT474 T798M cells, normalized to expression in GFP cells for each ligand. B . Lysates prepared from BT474 GFP (± 40 ng/ml TGFα) and BT474 T798M were immunoprecipitated with a HER2 C-terminal antibody followed by immunoblot analyses with the indicated antibodies. C . Lysates prepared from MCF10A WT (± 40 ng/ml TGFα) and MCF10A T798M were analyzed as in B. D . BT474 T798M cells were treated with 20 μg/ml cetuximab and 1 μM lapatinib, either alone or in combination for 5 days. MTT assays were performed at the end of treatment. E . Cells were treated with 20 μg/ml cetuximab and/or 1 μM lapatinib for 3 h. Lysates were prepared and subjected to immunoblot analyses with the indicated antibodies. F . For 3D Matrigel growth assays, cells were treated with 20 μg/ml trastuzumab, 20 μg/ml cetuximab or the combination. Cells were plated in duplicates and images captured on day 14. Fresh medium and inhibitors were replenished every 3 days. G . Average acinar area was quantified with the ImageJ software. Each bar represents the mean acinar area ± S.E.M ( n =2). H . Cells were treated with 20 μg/ml cetuximab and/or and 20 μg/ml trastuzumab and lysates analyzed as in E. I . BT474 T798M xenografts were established in female athymic mice as indicated in Methods. Once tumors reached at least ≥200 mm 3 in volume, mice were treated with trastuzumab (30 mg/kg i.p. twice per week), cetuximab (1 mg i.p. twice per week) or both antibodies. Each data point represents the mean tumor volume ± SEM. *, p

Techniques Used: Quantitative RT-PCR, Expressing, Immunoprecipitation, MTT Assay, Software, Mouse Assay

14) Product Images from "Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells"

Article Title: Synergistic Effect of MiR-146a Mimic and Cetuximab on Hepatocellular Carcinoma Cells

Journal: BioMed Research International

doi: 10.1155/2014/384121

Effect of combining miR-146a mimic and cetuximab on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a mimic and negative control (200 nM). Meanwhile, cetuximab was added and the cells were cultured up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; Cetu: cetuximab; Mimi: miR-146a mimic; comb: combination of cetuximab and miR-146a mimic.
Figure Legend Snippet: Effect of combining miR-146a mimic and cetuximab on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a mimic and negative control (200 nM). Meanwhile, cetuximab was added and the cells were cultured up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; Cetu: cetuximab; Mimi: miR-146a mimic; comb: combination of cetuximab and miR-146a mimic.

Techniques Used: Cell Culture, Transfection, Negative Control, Western Blot

Effect of miR-146a on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a inhibitor, miR-146a mimic, EGFR siRNA, and their negative controls (200 nM) up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; C1: negative control for miRNA inhibitor; Inhi: miR-146a inhibitor; C2: negative control for miRNA mimic; Mimi: miR-146a mimic; Si: EGFR siRNA.
Figure Legend Snippet: Effect of miR-146a on EGFR and its downstream pathway signals in HCC HepG2 cells. HepG2 cells (2.5 × 10 4 cells per well in 24-well plate) were cultured for 24 h then transfected with miR-146a inhibitor, miR-146a mimic, EGFR siRNA, and their negative controls (200 nM) up to another 96 h. Western blot and signal intensity of the bands were shown. Antibodies included phospho-EGFR (p-EGFR), total-EGFR (t-EGFR), p-ERK1/2, t-ERK1/2, p-AKT, t-AKT, p-stat5, t-stat5, and β -Actin. M: mock control; C1: negative control for miRNA inhibitor; Inhi: miR-146a inhibitor; C2: negative control for miRNA mimic; Mimi: miR-146a mimic; Si: EGFR siRNA.

Techniques Used: Cell Culture, Transfection, Western Blot, Negative Control

15) Product Images from "Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2"

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

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

doi: 10.1016/j.neo.2018.11.004

Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).
Figure Legend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

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

Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.
Figure Legend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

Techniques Used: Cell Culture, Transfection, WST-1 Assay, Western Blot

16) Product Images from "Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion"

Article Title: Elevated Expression of Fn14 in Non-Small Cell Lung Cancer Correlates with Activated EGFR and Promotes Tumor Cell Migration and Invasion

Journal: The American Journal of Pathology

doi: 10.1016/j.ajpath.2012.03.026

Erlotinib treatment of TKI-sensitive NSCLC cells decreases Fn14 expression. A: Serum-starved HCC827 cells containing the EGFR E746-A750–activating mutation were treated with vehicle for 12 hours or 1 μmol/L erlotinib for the indicated
Figure Legend Snippet: Erlotinib treatment of TKI-sensitive NSCLC cells decreases Fn14 expression. A: Serum-starved HCC827 cells containing the EGFR E746-A750–activating mutation were treated with vehicle for 12 hours or 1 μmol/L erlotinib for the indicated

Techniques Used: Expressing, Mutagenesis

17) Product Images from "Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway"

Article Title: Lupeol induces apoptosis and inhibits invasion in gallbladder carcinoma GBC-SD cells by suppression of EGFR/MMP-9 signaling pathway

Journal: Cytotechnology

doi: 10.1007/s10616-014-9763-7

Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification ×400). b – d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification ×400). e – g Expression of p-EGFR, MMP-9 and PCNA was quantified in percentages of positive cells within five medium-power fields under microscope and shown in histograms. Considered as * P
Figure Legend Snippet: Lupeol down-regulates p-EGFR, MMP-9 and PCNA in GBC-SD xenografts. a H E staining analyses of the pathological features of the tumors from the four groups (original magnification ×400). b – d The expression of p-EGFR, MMP-9 and PCNA in xenograft tumors was analyzed by immunohistochemistry (original magnification ×400). e – g Expression of p-EGFR, MMP-9 and PCNA was quantified in percentages of positive cells within five medium-power fields under microscope and shown in histograms. Considered as * P

Techniques Used: Staining, Expressing, Immunohistochemistry, Microscopy

Effect of lupeol on protein expression of EGFR family and MMP-9. a Expression of EGFR, p-EGFR, AKT, p-AKT and MMP-9 was analysed by western blot assay. GAPDH was used as the sample loading control. For one experiment, three assays were carried out but only one set of gels is shown. In GBC-SD cells treated with various lupeol concentrations for 36 h, the expression of EGFR and AKT did not show significant changes while p-EGFR, p-AKT and MMP-9 expression declined remarkably compared to the control group. b The expression of p-EGFR, p-AKT and MMP-9 protein levels in every group of GBC-SD cells was analyzed by densitometry normalized with the corresponding total AKT and ERK and GAPDH density with the ratios of p-AKT/AKT, p-ERK/ERK and MMP-9/GAPDH. Considered as * P
Figure Legend Snippet: Effect of lupeol on protein expression of EGFR family and MMP-9. a Expression of EGFR, p-EGFR, AKT, p-AKT and MMP-9 was analysed by western blot assay. GAPDH was used as the sample loading control. For one experiment, three assays were carried out but only one set of gels is shown. In GBC-SD cells treated with various lupeol concentrations for 36 h, the expression of EGFR and AKT did not show significant changes while p-EGFR, p-AKT and MMP-9 expression declined remarkably compared to the control group. b The expression of p-EGFR, p-AKT and MMP-9 protein levels in every group of GBC-SD cells was analyzed by densitometry normalized with the corresponding total AKT and ERK and GAPDH density with the ratios of p-AKT/AKT, p-ERK/ERK and MMP-9/GAPDH. Considered as * P

Techniques Used: Expressing, Western Blot

18) Product Images from "Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR"

Article Title: Conformations of tissue plasminogen activator (tPA) orchestrate neuronal survival by a crosstalk between EGFR and NMDAR

Journal: Cell Death & Disease

doi: 10.1038/cddis.2015.296

Both sc-tPA and tc-tPA at 300 nM can modulate EGFR signaling. ( a ) Representative immunoblots for phospho-EGFR ( A – C : tyrosine 1173; D – F : tyrosine 992) and total EGFR on neurons (12–13 DIV) after treatments with EGF (50 ng/ml), sc-tPA or tc-tPA (300 nM) during 15 min. ( B , C , E and F ) Quantifications of phosphorylated EGFRs and total EGFRs were compared with control (mean±S.E.M.; N =3 or 4 experiments; * P
Figure Legend Snippet: Both sc-tPA and tc-tPA at 300 nM can modulate EGFR signaling. ( a ) Representative immunoblots for phospho-EGFR ( A – C : tyrosine 1173; D – F : tyrosine 992) and total EGFR on neurons (12–13 DIV) after treatments with EGF (50 ng/ml), sc-tPA or tc-tPA (300 nM) during 15 min. ( B , C , E and F ) Quantifications of phosphorylated EGFRs and total EGFRs were compared with control (mean±S.E.M.; N =3 or 4 experiments; * P

Techniques Used: Western Blot

19) Product Images from "The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment *"

Article Title: The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.612754

Model for aggregate-mediated EGFR stabilization and anoikis suppression in ECM-detached ErbB2-overexpressing cells. A , model for aggregate-mediated ECM-detached survival in ErbB2-overexpressing cells. B , model for anoikis induction upon aggregate disruption
Figure Legend Snippet: Model for aggregate-mediated EGFR stabilization and anoikis suppression in ECM-detached ErbB2-overexpressing cells. A , model for aggregate-mediated ECM-detached survival in ErbB2-overexpressing cells. B , model for anoikis induction upon aggregate disruption

Techniques Used:

20) Product Images from "ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway"

Article Title: ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0798-z

Increased activation of ER-α36-mediated nongenomic estrogen signaling is responsible for cisplatin resistance. a , b MCF-7 and MCF-7/DDP cells ( a ) or MCF-7/V and MCF-7/ER-α36 cells ( b ) were harvested and the protein levels of ER-α36, EGFR, HER-2, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. c , d MCF-7/DDP ( c ) and MCF-7/ER-α36 ( d ) cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. Then the cells were collected and the levels of ER-α36, EGFR, HER-2, P-ERK, ERK were analyzed by western blot
Figure Legend Snippet: Increased activation of ER-α36-mediated nongenomic estrogen signaling is responsible for cisplatin resistance. a , b MCF-7 and MCF-7/DDP cells ( a ) or MCF-7/V and MCF-7/ER-α36 cells ( b ) were harvested and the protein levels of ER-α36, EGFR, HER-2, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. c , d MCF-7/DDP ( c ) and MCF-7/ER-α36 ( d ) cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. Then the cells were collected and the levels of ER-α36, EGFR, HER-2, P-ERK, ERK were analyzed by western blot

Techniques Used: Activation Assay, Western Blot, Transfection, Negative Control

Disruption of ER-α36-mediated nongenomic estrogen signaling increases cisplatin sensitivity in breast cancer cells. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the levels of ER-α36, total EGFR (EGFR) and phosphorylated EGFR (P-EGFR), total HER-2 (HER-2) and phosphorylated HER-2 (P-HER-2), total ERK (ERK) and phosphorylated ERK (P-ERK) were evaluated using western blot. b MCF-7 cells were treated as in ( a ), and then the cell proliferation was measured with CCK-8 assay kit. c MCF-7/ER-α36 cells were treated with 5 μg/mL cisplatin for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the total ERK (ERK) and phosphorylated ERK (P-ERK) was detected by western blot. d MCF-7/ER-α36 cells were treated as in ( c ), and then the cell proliferation was examined using CCK-8 assay kit. * P
Figure Legend Snippet: Disruption of ER-α36-mediated nongenomic estrogen signaling increases cisplatin sensitivity in breast cancer cells. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the levels of ER-α36, total EGFR (EGFR) and phosphorylated EGFR (P-EGFR), total HER-2 (HER-2) and phosphorylated HER-2 (P-HER-2), total ERK (ERK) and phosphorylated ERK (P-ERK) were evaluated using western blot. b MCF-7 cells were treated as in ( a ), and then the cell proliferation was measured with CCK-8 assay kit. c MCF-7/ER-α36 cells were treated with 5 μg/mL cisplatin for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the total ERK (ERK) and phosphorylated ERK (P-ERK) was detected by western blot. d MCF-7/ER-α36 cells were treated as in ( c ), and then the cell proliferation was examined using CCK-8 assay kit. * P

Techniques Used: Western Blot, CCK-8 Assay

Up-regulation of ER-α36 leads to increased activation of nongenomic estrogen signaling. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h. Then the protein levels of EGFR, HER-2, ER-α36, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. b , c The quantitative analysis of cisplatin-induced expression of ER-α36, EGFR, HER-2 and P-ERK/ERK of ( a ). d MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of EGFR, HER-2, ER-α36, ERK and P-ERK were analyzed by western blot. e The quantitative analysis of cisplatin-induced expression of P-ERK/ERK of ( d ). f MCF-7 cells were treated as in ( a ). The cell lysates were immunoprecipitated with anti-HER-2 or anti-EGFR antibodies. Then the immunoprecipitates were separated by SDS-PAGE and probed with anti-ER-α36 antibodies. Immunoprecipitation of IgG was used as a negative control
Figure Legend Snippet: Up-regulation of ER-α36 leads to increased activation of nongenomic estrogen signaling. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h. Then the protein levels of EGFR, HER-2, ER-α36, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. b , c The quantitative analysis of cisplatin-induced expression of ER-α36, EGFR, HER-2 and P-ERK/ERK of ( a ). d MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of EGFR, HER-2, ER-α36, ERK and P-ERK were analyzed by western blot. e The quantitative analysis of cisplatin-induced expression of P-ERK/ERK of ( d ). f MCF-7 cells were treated as in ( a ). The cell lysates were immunoprecipitated with anti-HER-2 or anti-EGFR antibodies. Then the immunoprecipitates were separated by SDS-PAGE and probed with anti-ER-α36 antibodies. Immunoprecipitation of IgG was used as a negative control

Techniques Used: Activation Assay, Western Blot, Expressing, Multiple Displacement Amplification, Immunoprecipitation, SDS Page, Negative Control

21) Product Images from "Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells"

Article Title: Uropathogenic Escherichia coli invades bladder epithelial cells by activating kinase networks in host cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.003499

Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p
Figure Legend Snippet: Role of EGFR, Akt, and mTORCs in UTI89 invasion of mouse bladder. A and B , strips of mouse transitional epithelial tissue were individually placed in wells (12-well plates) and treated or not with the indicated inhibitor for 1 h at 37 °C. UPEC (5 × 10 7 /ml) were added for additional 2 h, and tissue strips were washed three times with PBS. The tissue strips were lysed in PBS containing 0.1% (v/v) Triton X-100. For one part ( A ), cell lysates were analyzed for EGFR and Akt phosphorylation by Western blotting using the indicated antibodies. GAPDH was used as a protein loading control. For the second part ( B ), dilutions of lysates were plated on LB agar plates to allow colony growth. Colonies were counted, and data are presented as the -fold change of control values. Statistical analysis was performed by Student's t test compared with the nontreated ( NT ) control. *, p

Techniques Used: Western Blot

22) Product Images from "Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism"

Article Title: Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism

Journal: Oncogene

doi: 10.1038/onc.2015.353

A simplified scheme of the paracrine effects of estrogen in brain metastases Consistent with the notion that breast cancer cells benefit from normal homeostasis mechanisms at the metastatic site ( 48 , 50 ), our data supports a novel mechanism whereby brain metastatic cells exploit estrogen signaling though ER+ astrocytes. Estrogen can activate ERs on astrocytes, leading to transcriptional upregulation of EGFR ligands, activation of EGFR on TN EGFR+ brain metastatic breast cancer cells, and upregulation of several metastatic mediators, including S100A4. Given the complex bi-directional interactions between astrocytes and breast cancer cells, it is possible that other factors regulated by ERs in astrocytes and receptors on brain metastatic cells participate in this process. Ovarian and local estrogen depletion decrease brain metastatic colonization of TN EGFR+ 231BR cells in vivo , emphasizing the need to consider the brain microenvironment in designing novel therapies for brain metastases and the possibility to extend the use of aromatase inhibitors in the prevention of brain metastases in TN EGFR+ patients.
Figure Legend Snippet: A simplified scheme of the paracrine effects of estrogen in brain metastases Consistent with the notion that breast cancer cells benefit from normal homeostasis mechanisms at the metastatic site ( 48 , 50 ), our data supports a novel mechanism whereby brain metastatic cells exploit estrogen signaling though ER+ astrocytes. Estrogen can activate ERs on astrocytes, leading to transcriptional upregulation of EGFR ligands, activation of EGFR on TN EGFR+ brain metastatic breast cancer cells, and upregulation of several metastatic mediators, including S100A4. Given the complex bi-directional interactions between astrocytes and breast cancer cells, it is possible that other factors regulated by ERs in astrocytes and receptors on brain metastatic cells participate in this process. Ovarian and local estrogen depletion decrease brain metastatic colonization of TN EGFR+ 231BR cells in vivo , emphasizing the need to consider the brain microenvironment in designing novel therapies for brain metastases and the possibility to extend the use of aromatase inhibitors in the prevention of brain metastases in TN EGFR+ patients.

Techniques Used: Activation Assay, In Vivo

E2 upregulates EGFR ligands in astrocytes and results in EGFR activation in brain metastatic cells a. Primary mouse astrocytes were treated with vehicle (OH) or 10 nM E2 for the indicated times and qRT-PCR was used to measure Egf, Tgfα and Ereg mRNA levels. Bars represent fold change mRNA levels normalized to mouse β-Actin mRNA and relative to OH-treated. *P
Figure Legend Snippet: E2 upregulates EGFR ligands in astrocytes and results in EGFR activation in brain metastatic cells a. Primary mouse astrocytes were treated with vehicle (OH) or 10 nM E2 for the indicated times and qRT-PCR was used to measure Egf, Tgfα and Ereg mRNA levels. Bars represent fold change mRNA levels normalized to mouse β-Actin mRNA and relative to OH-treated. *P

Techniques Used: Activation Assay, Quantitative RT-PCR

23) Product Images from "Gefitinib sensitization of cisplatin-resistant wild-type EGFR non-small cell lung cancer cells"

Article Title: Gefitinib sensitization of cisplatin-resistant wild-type EGFR non-small cell lung cancer cells

Journal: Journal of Cancer Research and Clinical Oncology

doi: 10.1007/s00432-020-03228-4

Significant phosphorylation of EGFR, AKT, ERK in H358 R and A549 R cell lines. a Cells were fixed and incubated with p-EGFR antibody. Fluorescence was measured by flow cytometry. b Expression of p-EGFR, p-AKT, p-ERK in H358 R and A549 R cells by western blotting analysis (* P
Figure Legend Snippet: Significant phosphorylation of EGFR, AKT, ERK in H358 R and A549 R cell lines. a Cells were fixed and incubated with p-EGFR antibody. Fluorescence was measured by flow cytometry. b Expression of p-EGFR, p-AKT, p-ERK in H358 R and A549 R cells by western blotting analysis (* P

Techniques Used: Incubation, Fluorescence, Flow Cytometry, Expressing, Western Blot

Gefitinib (GFB) inhibited EGFR/AKT/ERK phosphorylation in H358 R and A549 R cells. a Detected EGFR/AKT/ERK phosphorylation levels after 48 h of gefitinib (GFB) at different intervention concentrations by western blot. The minimum concentration of gefitinib (GFB) inhibiting p-EGFR, p-AKT, p-ERK in H358 R is 5 μM. b Similarly, 10 μM gefitinib (GFB) could simultaneously inhibit p-EGFR, p-AKT, p-ERK in A549 R cells
Figure Legend Snippet: Gefitinib (GFB) inhibited EGFR/AKT/ERK phosphorylation in H358 R and A549 R cells. a Detected EGFR/AKT/ERK phosphorylation levels after 48 h of gefitinib (GFB) at different intervention concentrations by western blot. The minimum concentration of gefitinib (GFB) inhibiting p-EGFR, p-AKT, p-ERK in H358 R is 5 μM. b Similarly, 10 μM gefitinib (GFB) could simultaneously inhibit p-EGFR, p-AKT, p-ERK in A549 R cells

Techniques Used: Western Blot, Concentration Assay

24) Product Images from "Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2"

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

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

doi: 10.1016/j.neo.2018.11.004

Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).
Figure Legend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

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

Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.
Figure Legend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

Techniques Used: Cell Culture, Transfection, WST-1 Assay, Western Blot

25) Product Images from "Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins"

Article Title: Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins

Journal: PLoS ONE

doi: 10.1371/journal.pone.0158116

Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of total p53 protein expression in 2D- and 3D-cultured LNCaP cells. Bars represent the relative protein quantification of p53 isoforms at ~144, ~100, ~58, and ~44 kda on the basis of β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05 using one-way ANOVA and unpaired t-tests.
Figure Legend Snippet: Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of total p53 protein expression in 2D- and 3D-cultured LNCaP cells. Bars represent the relative protein quantification of p53 isoforms at ~144, ~100, ~58, and ~44 kda on the basis of β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05 using one-way ANOVA and unpaired t-tests.

Techniques Used: Expressing, Cell Culture

Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of the expression of (A) total EGFR and (B) β-III tubulin in 2D- and 3D-cultured DU145 cells. Bars represent the relative protein quantification of (A) total EGFR and (B) β-III tubulin on the basis of GAPDH and β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05, **p≤0.01, and ***p≤0.001, using one-way ANOVA and unpaired t-tests.
Figure Legend Snippet: Total EGFR and β-III tubulin protein expression in DU145 cells. A representative image of the expression of (A) total EGFR and (B) β-III tubulin in 2D- and 3D-cultured DU145 cells. Bars represent the relative protein quantification of (A) total EGFR and (B) β-III tubulin on the basis of GAPDH and β-actin, and indicate the mean ± SEM (n = 3 for each group). *p≤0.05, **p≤0.01, and ***p≤0.001, using one-way ANOVA and unpaired t-tests.

Techniques Used: Expressing, Cell Culture

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

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

Journal: Gut Microbes

doi: 10.1080/19490976.2018.1479625

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

Techniques Used:

27) Product Images from "Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model"

Article Title: Antitumor and antiangiogenic effect of the dual EGFR and HER-2 tyrosine kinase inhibitor lapatinib in a lung cancer model

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-188

Intracellular signaling changes induced by lapatinib in A549 cells, analyzed by western blot . A . Immunoblots showing decreased levels of p-EGFR and p-HER-2 after stimulation with 100 ng/ml EGF and treatment with lapatinib. Downstream targets p-AKT, p-ERK1/2, c-Myc and PCNA were also reduced upon exposure to the drug. B . After lapatinib treatment, the proapoptotic protein Bak-1 was increased, the levels of the antiapoptotic proteins IAP-2 and Bcl-xL were reduced, and PARP was cleaved, thus demonstrating that the apoptotic pathway is switched on by this drug in A549 lung cancer cells.
Figure Legend Snippet: Intracellular signaling changes induced by lapatinib in A549 cells, analyzed by western blot . A . Immunoblots showing decreased levels of p-EGFR and p-HER-2 after stimulation with 100 ng/ml EGF and treatment with lapatinib. Downstream targets p-AKT, p-ERK1/2, c-Myc and PCNA were also reduced upon exposure to the drug. B . After lapatinib treatment, the proapoptotic protein Bak-1 was increased, the levels of the antiapoptotic proteins IAP-2 and Bcl-xL were reduced, and PARP was cleaved, thus demonstrating that the apoptotic pathway is switched on by this drug in A549 lung cancer cells.

Techniques Used: Western Blot

28) Product Images from "Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells"

Article Title: Gefitinib resistance resulted from STAT3-mediated Akt activation in lung cancer cells

Journal: Oncotarget

doi:

Gefitinib promotes EGFR-STAT3 interaction (A) Immunoprecipitation assay (left pannel) demonstrates direct physical binding of EGFR and STAT3 induced by gefitinib treatment. Cells were treated with 8μM gefitinib for 6 hours. The samples were precipitated with STAT3 antibody and detected with antibodies against EGFR and STAT3 sequentially. (B) Immunoblotting analysis shows the effect of gefitinib on SOCS1 and SOCS3 in A549 cells.
Figure Legend Snippet: Gefitinib promotes EGFR-STAT3 interaction (A) Immunoprecipitation assay (left pannel) demonstrates direct physical binding of EGFR and STAT3 induced by gefitinib treatment. Cells were treated with 8μM gefitinib for 6 hours. The samples were precipitated with STAT3 antibody and detected with antibodies against EGFR and STAT3 sequentially. (B) Immunoblotting analysis shows the effect of gefitinib on SOCS1 and SOCS3 in A549 cells.

Techniques Used: Immunoprecipitation, Binding Assay

Gefitinib inhibits EGFR constitutively and substantially (A) Immunofluorescence test shows the process of internalization of EGFR at proceeding time points after treatment of gefitinib in A549 cells. (B) Gefitinib treatment induced continuous inhibition of EGFR phosphorylation on tyrosine 1068 (Y1068) and threonine 669 (T669) without recovery at the later time points. (C) Semi-quantification of Akt recovery following gefitinib treatment.
Figure Legend Snippet: Gefitinib inhibits EGFR constitutively and substantially (A) Immunofluorescence test shows the process of internalization of EGFR at proceeding time points after treatment of gefitinib in A549 cells. (B) Gefitinib treatment induced continuous inhibition of EGFR phosphorylation on tyrosine 1068 (Y1068) and threonine 669 (T669) without recovery at the later time points. (C) Semi-quantification of Akt recovery following gefitinib treatment.

Techniques Used: Immunofluorescence, Inhibition

Gefitinib inhibits EGFR phosphorylation but induces STAT3 activation in lung caner cells Cells were subject to time-dependent treatment of gefitinib at 4μM (left panel) and 8μM (right panel) for the indicated time periods. The expression and activity of EGFR, Akt and STAT3 in A549 cells were determined by Western Blot.
Figure Legend Snippet: Gefitinib inhibits EGFR phosphorylation but induces STAT3 activation in lung caner cells Cells were subject to time-dependent treatment of gefitinib at 4μM (left panel) and 8μM (right panel) for the indicated time periods. The expression and activity of EGFR, Akt and STAT3 in A549 cells were determined by Western Blot.

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

Chemical inhibitor and gene silencing of STAT3 suppresses succeeding recovery of Akt activation after gefitinib treatment (A B) Immunoblotting analysis of expressions and activities of EGFR, Akt, STAT3, ERK, and P38 under time-dependent treatment with 4μM (left panel) and 8μM (right panel) gefitinib combined with or without 100 μM Stattic (STAT3 inhibitor) for up to 6 hours in A549 cells. (C) Silencing STAT3 by siRNA diminishes gefitinib-induced Akt recovery in A549 cells. (D) Semi-quantification of the Akt S473 phosphorylation in the cells treated with gefitinib and transfected with control siRNA (siCtrl, left panel) or STAT3 siRNA (siSTAT3, right panel).
Figure Legend Snippet: Chemical inhibitor and gene silencing of STAT3 suppresses succeeding recovery of Akt activation after gefitinib treatment (A B) Immunoblotting analysis of expressions and activities of EGFR, Akt, STAT3, ERK, and P38 under time-dependent treatment with 4μM (left panel) and 8μM (right panel) gefitinib combined with or without 100 μM Stattic (STAT3 inhibitor) for up to 6 hours in A549 cells. (C) Silencing STAT3 by siRNA diminishes gefitinib-induced Akt recovery in A549 cells. (D) Semi-quantification of the Akt S473 phosphorylation in the cells treated with gefitinib and transfected with control siRNA (siCtrl, left panel) or STAT3 siRNA (siSTAT3, right panel).

Techniques Used: Activation Assay, Transfection

Gefitinib treatment on Akt inhibition and recovery (A) Westernblotting analysis shows that gefitinib inhibits multiple protein regulators involved in EGFR signaling pathway and induces Akt recovery in A549 cells (human lung cancer cell line). The cells were either untreated (blank, BLK) or treated with 4 or 8 µM gefitinib for 0.5 to 6 h. The recovery at the later time points of gefitinib treatment can be seen only for Akt, but not PI3K, p38, JNK, and ERK. (B) Westernbloting data showed Akt recovery in NCI-H2023 cells and NCI-H2126 cells treated with gefitinib for the indicated hours (h).
Figure Legend Snippet: Gefitinib treatment on Akt inhibition and recovery (A) Westernblotting analysis shows that gefitinib inhibits multiple protein regulators involved in EGFR signaling pathway and induces Akt recovery in A549 cells (human lung cancer cell line). The cells were either untreated (blank, BLK) or treated with 4 or 8 µM gefitinib for 0.5 to 6 h. The recovery at the later time points of gefitinib treatment can be seen only for Akt, but not PI3K, p38, JNK, and ERK. (B) Westernbloting data showed Akt recovery in NCI-H2023 cells and NCI-H2126 cells treated with gefitinib for the indicated hours (h).

Techniques Used: Inhibition

29) Product Images from "A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation"

Article Title: A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation

Journal: Journal of Cell Science

doi: 10.1242/jcs.128280

AT 1 R–EGFR transactivation occurs in HMEC-LST-AT 1 R cells. (A) HMEC-LST cells (mCherry or AT 1 R transduced) were serum starved then stimulated with 100 nM AngII for 5-10 minutes. Robust activation of the EGFR (pY1068) and ERK1/2 was
Figure Legend Snippet: AT 1 R–EGFR transactivation occurs in HMEC-LST-AT 1 R cells. (A) HMEC-LST cells (mCherry or AT 1 R transduced) were serum starved then stimulated with 100 nM AngII for 5-10 minutes. Robust activation of the EGFR (pY1068) and ERK1/2 was

Techniques Used: Activation Assay

30) Product Images from "Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non‐small‐cell lung cancer cell lines), Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non‐small‐cell lung cancer cell lines"

Article Title: Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non‐small‐cell lung cancer cell lines), Tivantinib (ARQ 197) efficacy is independent of MET inhibition in non‐small‐cell lung cancer cell lines

Journal: Molecular Oncology

doi: 10.1016/j.molonc.2014.08.011

HCC827 GR6 cells are sensitive to tivantinib and resistant to crizotinib or PHA‐665752 alone, but combined MET and EGFR inhibition blocks PI3K/AKT and ERK signaling and restores sensitivity. (A) HCC827 GR6 cells treated with increasing concentrations
Figure Legend Snippet: HCC827 GR6 cells are sensitive to tivantinib and resistant to crizotinib or PHA‐665752 alone, but combined MET and EGFR inhibition blocks PI3K/AKT and ERK signaling and restores sensitivity. (A) HCC827 GR6 cells treated with increasing concentrations

Techniques Used: Inhibition

31) Product Images from "Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models"

Article Title: Therapeutic potential of combined BRAF/MEK blockade in BRAF-wild type preclinical tumor models

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0820-5

Working model of intra-pathway feedbacks and BRAF/MEK growth-inhibitory synergism. a In BRAF-wt/KRAS-mut contexts, selective BRAF inhibition induces BRAF-CRAF dimerization, which hyperactivates the MAPK pathway, thus resulting in relative resistance to treatment. In BRAF-wt/KRAS-wt contexts, paradoxical MAPK activation may be sustained by the RAS-dependent upstream signaling of RTKs (in particular EGFR family members). b Upon allosteric MEK inhibition, the MAPK pathway downstream of a mutant KRAS is efficiently shut down; however, MEK inhibition-induced removal of ERK-mediated feedback RTK inhibition may result in incomplete MAPK pathway inhibition or pathway reactivation, again resulting in relative resistance to the drug. c Combined BRAF/MEK inhibition results in efficient pathway blockade and a synergistic effect on cell growth inhibition, particularly downstream of a mutant KRAS; however, as highlighted also in panel b, in KRAS-wt contexts removal of ERK-mediated feedback RTK inhibition may result in RTK-dependent pathway (re)activation, thus resulting in only partial blockade of downstream signaling. d Thus, in KRAS-wt contexts, triple RTK (EGFR family in the specific case discussed here)/BRAF/MEK inhibition is hypothesized to completely prevent paradoxical MAPK activation; functional growth-inhibitory synergism will then vary according to the degree of intrinsic sensitivity/resistance to RTK inhibition
Figure Legend Snippet: Working model of intra-pathway feedbacks and BRAF/MEK growth-inhibitory synergism. a In BRAF-wt/KRAS-mut contexts, selective BRAF inhibition induces BRAF-CRAF dimerization, which hyperactivates the MAPK pathway, thus resulting in relative resistance to treatment. In BRAF-wt/KRAS-wt contexts, paradoxical MAPK activation may be sustained by the RAS-dependent upstream signaling of RTKs (in particular EGFR family members). b Upon allosteric MEK inhibition, the MAPK pathway downstream of a mutant KRAS is efficiently shut down; however, MEK inhibition-induced removal of ERK-mediated feedback RTK inhibition may result in incomplete MAPK pathway inhibition or pathway reactivation, again resulting in relative resistance to the drug. c Combined BRAF/MEK inhibition results in efficient pathway blockade and a synergistic effect on cell growth inhibition, particularly downstream of a mutant KRAS; however, as highlighted also in panel b, in KRAS-wt contexts removal of ERK-mediated feedback RTK inhibition may result in RTK-dependent pathway (re)activation, thus resulting in only partial blockade of downstream signaling. d Thus, in KRAS-wt contexts, triple RTK (EGFR family in the specific case discussed here)/BRAF/MEK inhibition is hypothesized to completely prevent paradoxical MAPK activation; functional growth-inhibitory synergism will then vary according to the degree of intrinsic sensitivity/resistance to RTK inhibition

Techniques Used: Inhibition, Activation Assay, Mutagenesis, Functional Assay

Selective BRAF inhibition induces EGFR family-dependent MAPK hyperactivation in Calu-3 cells. a and b The NSCLC cell line Calu-3 ( HER2 -amplified, KRAS -wt) was treated with increasing concentrations of dabrafenib (0.01–10 μM) alone a or in combination with trametinib (ratio 1:1000; b ) for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the proteins indicated. Western blot with Hsp70 specific antibody is shown as protein loading and blotting control. c Calu-3 cells were treated with increasing concentrations of dabrafenib (0.01–10 μM) and trametinib (0.01 nM–10 nM) alone or in combination for 72 h. Cell viability was assessed by Crystal violet assay and pharmacologic interactions were evaluated using the Calcusyn software. The results represent the average ± SD of three independent experiments. d Calu-3 and HCC827 ( EGFR -mut) cells were treated with increasing concentrations of dabrafenib (0.1–10 μM) and lapatinib (0.1–10 μM) for 4 h. The proteins were subjected to Western Blotting and analyzed for the indicated antibodies. e MiaPaCa2, A549 and A427 cells were treated with dabrafenib (10 μM) and lapatinib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated
Figure Legend Snippet: Selective BRAF inhibition induces EGFR family-dependent MAPK hyperactivation in Calu-3 cells. a and b The NSCLC cell line Calu-3 ( HER2 -amplified, KRAS -wt) was treated with increasing concentrations of dabrafenib (0.01–10 μM) alone a or in combination with trametinib (ratio 1:1000; b ) for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the proteins indicated. Western blot with Hsp70 specific antibody is shown as protein loading and blotting control. c Calu-3 cells were treated with increasing concentrations of dabrafenib (0.01–10 μM) and trametinib (0.01 nM–10 nM) alone or in combination for 72 h. Cell viability was assessed by Crystal violet assay and pharmacologic interactions were evaluated using the Calcusyn software. The results represent the average ± SD of three independent experiments. d Calu-3 and HCC827 ( EGFR -mut) cells were treated with increasing concentrations of dabrafenib (0.1–10 μM) and lapatinib (0.1–10 μM) for 4 h. The proteins were subjected to Western Blotting and analyzed for the indicated antibodies. e MiaPaCa2, A549 and A427 cells were treated with dabrafenib (10 μM) and lapatinib (10 μM) alone or in combination for 4 h. The cells were lysed and analyzed by Western Blotting using antibodies specific for the protein above indicated

Techniques Used: Inhibition, Amplification, Western Blot, Crystal Violet Assay, Software

32) Product Images from "Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers"

Article Title: Dual targeting of HER3 and MEK may overcome HER3-dependent drug-resistance of colon cancers

Journal: Oncotarget

doi: 10.18632/oncotarget.11400

Patritumab administration inhibits the PI3K survival pathway and activates the MAPK proliferation pathway Total cell lysates from HT29 ( A ), Pt-1 ( B ), Pt-2 ( C ), and Pt-3 ( D ) cells, incubated with 10 ng/ml HRG-β1 and 10 μg/ml patritumab antibody for the indicated times, were analyzed by immunoblot to evaluate the expression of total and P-HER3, HER2, EGFR, total and p-AKT, total and p-ERK. The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane.
Figure Legend Snippet: Patritumab administration inhibits the PI3K survival pathway and activates the MAPK proliferation pathway Total cell lysates from HT29 ( A ), Pt-1 ( B ), Pt-2 ( C ), and Pt-3 ( D ) cells, incubated with 10 ng/ml HRG-β1 and 10 μg/ml patritumab antibody for the indicated times, were analyzed by immunoblot to evaluate the expression of total and P-HER3, HER2, EGFR, total and p-AKT, total and p-ERK. The anti-HSP70 antibody was used to validate equivalent amount of loaded proteins in each lane.

Techniques Used: Incubation, Expressing

33) Product Images from "Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation"

Article Title: Anti-inflammatory effect of afatinib (an EGFR-TKI) on OGD-induced neuroinflammation

Journal: Scientific Reports

doi: 10.1038/s41598-019-38676-7

Afatinib blocked OGD-induced EGFR activation and downstream signalings. ( A , B ) CTX-TNA2 cells were exposed to oxygen-glucose deprivation (OGD) for various durations (3, 6, 12 h). Western blot assay was employed to measure ( A ) the levels of phospho-EGFR (pEGFR) and total-EGFR (EGFR) as well as ( B ) phospho-ERK (pERK), total-ERK (ERK), phospho-AKT (pAKT), total-AKT (AKT) and β-Actin. Each lane contained 40 μg protein for all experiments. Graphs show statistical results of pEGFR and EGFR ( A ) as well as pERK, ERK, pAKT and AKT ( B ) from relative optical density of bands on the blots. *P
Figure Legend Snippet: Afatinib blocked OGD-induced EGFR activation and downstream signalings. ( A , B ) CTX-TNA2 cells were exposed to oxygen-glucose deprivation (OGD) for various durations (3, 6, 12 h). Western blot assay was employed to measure ( A ) the levels of phospho-EGFR (pEGFR) and total-EGFR (EGFR) as well as ( B ) phospho-ERK (pERK), total-ERK (ERK), phospho-AKT (pAKT), total-AKT (AKT) and β-Actin. Each lane contained 40 μg protein for all experiments. Graphs show statistical results of pEGFR and EGFR ( A ) as well as pERK, ERK, pAKT and AKT ( B ) from relative optical density of bands on the blots. *P

Techniques Used: Activation Assay, Western Blot

34) Product Images from "RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells"

Article Title: RIP1 Is a Novel Component of γ-ionizing Radiation-Induced Invasion of Non-Small Cell Lung Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21134584

IR induces RIP1 expression and activates epithelial-mesenchymal transition (EMT) in vitro/in vivo. ( A ) Lysates from A549 cells treated with IR (10 Gy) were subjected to immunoblotting to examine the expression levels of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3 p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( B ) A549 cells were exposed to IR (10 Gy) and treated with gefitinib (EGFR inhibitor, 10 µM). Immunoblotting was applied to examine the expression of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3, p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( C , D ) A549 cells subjected to IR (10 Gy) were treated with Src (20 µM) or STAT3 inhibitor (20 µM) for the indicated periods and Western blot analyses for RIP1, vimentin, MMP-2, MMP-9, Src, p-Src, STAT3, p-STAT3 and β-actin (loading control) were conducted. Data are representative of triplicate experiments. (***; p
Figure Legend Snippet: IR induces RIP1 expression and activates epithelial-mesenchymal transition (EMT) in vitro/in vivo. ( A ) Lysates from A549 cells treated with IR (10 Gy) were subjected to immunoblotting to examine the expression levels of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3 p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( B ) A549 cells were exposed to IR (10 Gy) and treated with gefitinib (EGFR inhibitor, 10 µM). Immunoblotting was applied to examine the expression of RIP1, MMP-2, MMP-9, EGFR, p-EGFR, Src, p-Src, STAT3, p-STAT3, vimentin and β-actin (loading control). Representative data from triplicate experiments are shown. ( C , D ) A549 cells subjected to IR (10 Gy) were treated with Src (20 µM) or STAT3 inhibitor (20 µM) for the indicated periods and Western blot analyses for RIP1, vimentin, MMP-2, MMP-9, Src, p-Src, STAT3, p-STAT3 and β-actin (loading control) were conducted. Data are representative of triplicate experiments. (***; p

Techniques Used: Expressing, In Vitro, In Vivo, Western Blot

Scheme of the potential IR-induced EGFR/NF-κB–RIP1–Src–STAT3–EMT pathway.
Figure Legend Snippet: Scheme of the potential IR-induced EGFR/NF-κB–RIP1–Src–STAT3–EMT pathway.

Techniques Used:

35) Product Images from "Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 MimeticsInduction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung AdenocarcinomasBIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR MutationsWhy Do Cancer Cells Become "Addicted" to Oncogenic Epidermal Growth Factor Receptor?"

Article Title: Gefitinib-Induced Killing of NSCLC Cell Lines Expressing Mutant EGFR Requires BIM and Can Be Enhanced by BH3 MimeticsInduction of BIM Is Essential for Apoptosis Triggered by EGFR Kinase Inhibitors in Mutant EGFR-Dependent Lung AdenocarcinomasBIM Mediates EGFR Tyrosine Kinase Inhibitor-Induced Apoptosis in Lung Cancers with Oncogenic EGFR MutationsWhy Do Cancer Cells Become "Addicted" to Oncogenic Epidermal Growth Factor Receptor?

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0040316

Effect of Gefitinib on NSCLC Cells Expressing WT or Mutant EGFR NSCLC cells expressing WT (H358) or mutant (HCC827, H1975, H1650, H3255) EGFR were treated with varying concentrations of gefitinib for 24 h (A) or 72 h (B). Cells were then either assessed for the phosphorylation status of ERK (A) or cell death (B). Western blotting (A) was performed to determine the phosphorylation status of ERK1/2 before and after treatment with gefitinib. An actin loading control is also shown. Cell death was assessed by Annexin V-FITC plus PI staining (B). Results represent mean ± standard error of the mean (SEM) of at least three experiments. NT, no treatment.
Figure Legend Snippet: Effect of Gefitinib on NSCLC Cells Expressing WT or Mutant EGFR NSCLC cells expressing WT (H358) or mutant (HCC827, H1975, H1650, H3255) EGFR were treated with varying concentrations of gefitinib for 24 h (A) or 72 h (B). Cells were then either assessed for the phosphorylation status of ERK (A) or cell death (B). Western blotting (A) was performed to determine the phosphorylation status of ERK1/2 before and after treatment with gefitinib. An actin loading control is also shown. Cell death was assessed by Annexin V-FITC plus PI staining (B). Results represent mean ± standard error of the mean (SEM) of at least three experiments. NT, no treatment.

Techniques Used: Expressing, Mutagenesis, Western Blot, Staining

36) Product Images from "Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors"

Article Title: Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors

Journal: Nature Communications

doi: 10.1038/s41467-018-07078-0

Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)
Figure Legend Snippet: Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)

Techniques Used: Mutagenesis, Binding Assay

EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)
Figure Legend Snippet: EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)

Techniques Used: In Vitro, In Vivo, Plasmid Preparation, Expressing, Mouse Assay, Injection, Two Tailed Test, Staining

Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown
Figure Legend Snippet: Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown

Techniques Used: Fluorescence, Plasmid Preparation, Expressing, Mutagenesis

37) Product Images from "Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors"

Article Title: Overcoming EGFRG724S-mediated osimertinib resistance through unique binding characteristics of second-generation EGFR inhibitors

Journal: Nature Communications

doi: 10.1038/s41467-018-07078-0

Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)
Figure Legend Snippet: Kinetic evaluation of second- and third-generation EGFR TKIs against EGFR mutant proteins. a Schematic overview of two-step binding mechanism of covalent inhibitors to kinases with K i (quotient of k off and k on ) describing the reversible binding affinity and k inact describing the rate of inactivation. b Time-dependent IC 50 -determination of afatinib and osimertinib on EGFR mutant proteins. Representative curves of single measurements in duplicates are shown. c Heatmap of biochemical IC 50 -, K i -, and k inact determination of second- and third-generation EGFR TKIs against EGFR mutant proteins. Values are the mean of three independent measurements in duplicates. d Immunoblotting results of NIH-3T3 cells (EGFR 19del or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR. Cells were treated for indicated times (0, 1, 3, 6, and 24 h) with osimertinib (1 µM) or afatinib (1 µM). HSP90 was used as loading control ( n = 3)

Techniques Used: Mutagenesis, Binding Assay

EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)
Figure Legend Snippet: EGFR G724S mediates resistance to third-generation EGFR inhibitors in vitro and in vivo. a Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S , or EGFR 19del+G724S ) monitoring phospho-EGFR and total EGFR under erlotinib treatment (24 h). HSP90 was used as loading control. b Immunoblotting of NIH-3T3 cells under osimertinib treatment (24 h) is shown. Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with c erlotinib or d osimertinib. Experiments were performed at least three times. e , f Graphs show tumor volume of mice injected with NIH-3T3 EGFR 19del and EGFR 19del+G724S cells treated with osimertinib (5 mg/kg, i.p., once daily). e Tumor volumes ( EGFR 19del vehicle, n = 7 mice; EGFR 19del osimertinib, n = 8 mice; EGFR 19del+G724S vehicle, n = 7 mice; EGFR 19del+G724S osimertinib, n =10 mice) were assessed for 20 days by longitudinal caliper measurements every second day following treatment initiation. f Tumor volumes were quantified after 8 days of treatment. Volume changes in the osimertinib treatment cohort (dark gray and green) were compared with the vehicle-treated control group (light gray and green). Each dot represents a single tumor per mouse. Significance is calculated by two-tailed Student’s t test, n.s.: non-significant. g Representative images of Cleaved Caspase-3 stainings. Tumors of mice bearing NIH-3T3 EGFR 19del or EGFR 19del+G724S cells were treated with vehicle solution HPMC (0, 5%) or osimertinib. Scale bar 100 μm. h Quantification of Cleaved Caspase-3 staining. Number of apoptotic cells in the osimertinib-treated cohort (dark gray and green) was compared with the vehicle-treated control group (light gray and green)

Techniques Used: In Vitro, In Vivo, Plasmid Preparation, Expressing, Mouse Assay, Injection, Two Tailed Test, Staining

Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown
Figure Legend Snippet: Biochemical profiling of EGFR G724S . a Homogeneous time-resolved fluorescence (HTRF) is used for IC 50 -determination for EGFR 19del and EGFR 19del +G724S with representative inhibitors. Representative dose–response curves of a single measurement in duplicates are shown. b Comparison of biochemical IC 50 -values with HTRF for the three generations of EGFR TKIs against EGFR 19del+G724S . Values are the mean of three independent measurements in duplicates. c Immunoblotting results of NIH-3T3 cells (empty vector, EGFR 19del , EGFR G724S or EGFR 19del+G724S ) showing phospho-EGFR and total EGFR under afatinib treatment (24 h). HSP90 was used as loading control ( n = 3). d Dose–response measurement of Ba/F3 cells expressing EGFR 19del , EGFR G724S , or EGFR 19del+G724S treated for 72 h with afatinib. Experiments were accomplished for at least three times. e Structure of exon 20 mutant EGFR , bound to 4-anilinoquinazoline based TKI PD168393, is shown

Techniques Used: Fluorescence, Plasmid Preparation, Expressing, Mutagenesis

38) Product Images from "Identification of Mutant K-Ras-dependent Phenotypes Using a Panel of Isogenic Cell Lines *"

Article Title: Identification of Mutant K-Ras-dependent Phenotypes Using a Panel of Isogenic Cell Lines *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.394130

Mutant KRas negatively regulates EGFR signaling. A , Hec1A cells deleted for either WT or mutant KRas were serum-starved or starved and stimulated with 100 ng/ml EGF for 10 min. 1 mg of lysate was reduced, alkylated, digested with Lys-C, and used to probe antibody microarrays. Signal for EGFR and Cbl phosphorylation (mean ± S.D. ( error bars , n = 3)) is shown. B , the indicated cell lines were cultured in 10% FBS, 0% FBS, or stimulated with 100 ng/ml EGF, and 40 μg of protein lysate was separated by SDS-PAGE and probed by Western blotting using the antibodies shown. C , results are the same as for B but using HMECs of the indicated genotypes.
Figure Legend Snippet: Mutant KRas negatively regulates EGFR signaling. A , Hec1A cells deleted for either WT or mutant KRas were serum-starved or starved and stimulated with 100 ng/ml EGF for 10 min. 1 mg of lysate was reduced, alkylated, digested with Lys-C, and used to probe antibody microarrays. Signal for EGFR and Cbl phosphorylation (mean ± S.D. ( error bars , n = 3)) is shown. B , the indicated cell lines were cultured in 10% FBS, 0% FBS, or stimulated with 100 ng/ml EGF, and 40 μg of protein lysate was separated by SDS-PAGE and probed by Western blotting using the antibodies shown. C , results are the same as for B but using HMECs of the indicated genotypes.

Techniques Used: Mutagenesis, Cell Culture, SDS Page, Western Blot

39) Product Images from "Cancer-Associated Fibroblasts derived from EGFR-TKI resistant tumors reverse EGFR pathway inhibition by EGFR-TKIs"

Article Title: Cancer-Associated Fibroblasts derived from EGFR-TKI resistant tumors reverse EGFR pathway inhibition by EGFR-TKIs

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-09-0460

GR CAFs modulate the EGFR-TKI response in co-cultured tumor cells. A B , Immunoblots for pEGFR, pMAPK and pAkt in GP epithelial tumor cells ( A ) and HCC827 cells ( B ) demonstrating effect of gefitinib treatment upon co-culture with GP or GR CAFs. Densitometric intensities for each marker were determined and the marker that demonstrates the most dramatic modification in each cell type in presence of gefitinib is presented.
Figure Legend Snippet: GR CAFs modulate the EGFR-TKI response in co-cultured tumor cells. A B , Immunoblots for pEGFR, pMAPK and pAkt in GP epithelial tumor cells ( A ) and HCC827 cells ( B ) demonstrating effect of gefitinib treatment upon co-culture with GP or GR CAFs. Densitometric intensities for each marker were determined and the marker that demonstrates the most dramatic modification in each cell type in presence of gefitinib is presented.

Techniques Used: Cell Culture, Western Blot, Co-Culture Assay, Marker, Modification

A , GP and GR epithelial tumor cells are equally responsive to gefitinib. Equal numbers of GP and GR epithelial tumor cells seeded in 96-well plates were treated in quadruplicates with increasing concentrations of gefitinib (0, 0.1, 1, 5, 10 and 20 µM) for 5 days. End point cell viability was measured using the MTS assay and the data are represented as a percentage of the untreated sample. B , Same as in A, except cells from 60mm dishes were lysed and analyzed by immunoblot for pEGFR, EGFR, pMAPK, and β-actin. C , h EMP-1 is expressed in the GR CAFs. Quantitative RT-PCR analysis of h EMP-1 mRNA comparing GP to GR in the tumor tissue, epithelial tumor cells, and CAFs. Data are normalized to GAPDH and presented as fold induction of GP tumor tissue. D , FISH analysis of purified GP and GR CAFs with CEP Y (DYZ1) probe ( green fluorescent signal ) depicting human nuclei ( white arrow ) and murine nuclei ( red arrow ). Nuclear counter staining with DAPI ( blue ).
Figure Legend Snippet: A , GP and GR epithelial tumor cells are equally responsive to gefitinib. Equal numbers of GP and GR epithelial tumor cells seeded in 96-well plates were treated in quadruplicates with increasing concentrations of gefitinib (0, 0.1, 1, 5, 10 and 20 µM) for 5 days. End point cell viability was measured using the MTS assay and the data are represented as a percentage of the untreated sample. B , Same as in A, except cells from 60mm dishes were lysed and analyzed by immunoblot for pEGFR, EGFR, pMAPK, and β-actin. C , h EMP-1 is expressed in the GR CAFs. Quantitative RT-PCR analysis of h EMP-1 mRNA comparing GP to GR in the tumor tissue, epithelial tumor cells, and CAFs. Data are normalized to GAPDH and presented as fold induction of GP tumor tissue. D , FISH analysis of purified GP and GR CAFs with CEP Y (DYZ1) probe ( green fluorescent signal ) depicting human nuclei ( white arrow ) and murine nuclei ( red arrow ). Nuclear counter staining with DAPI ( blue ).

Techniques Used: MTS Assay, Quantitative RT-PCR, Fluorescence In Situ Hybridization, Purification, Staining

40) Product Images from "ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway"

Article Title: ER-α36 mediates cisplatin resistance in breast cancer cells through EGFR/HER-2/ERK signaling pathway

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0798-z

Increased activation of ER-α36-mediated nongenomic estrogen signaling is responsible for cisplatin resistance. a , b MCF-7 and MCF-7/DDP cells ( a ) or MCF-7/V and MCF-7/ER-α36 cells ( b ) were harvested and the protein levels of ER-α36, EGFR, HER-2, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. c , d MCF-7/DDP ( c ) and MCF-7/ER-α36 ( d ) cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. Then the cells were collected and the levels of ER-α36, EGFR, HER-2, P-ERK, ERK were analyzed by western blot
Figure Legend Snippet: Increased activation of ER-α36-mediated nongenomic estrogen signaling is responsible for cisplatin resistance. a , b MCF-7 and MCF-7/DDP cells ( a ) or MCF-7/V and MCF-7/ER-α36 cells ( b ) were harvested and the protein levels of ER-α36, EGFR, HER-2, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. c , d MCF-7/DDP ( c ) and MCF-7/ER-α36 ( d ) cells were transfected with siER-α36 and negative control siRNA (siNC) for 48 h. Then the cells were collected and the levels of ER-α36, EGFR, HER-2, P-ERK, ERK were analyzed by western blot

Techniques Used: Activation Assay, Western Blot, Transfection, Negative Control

Disruption of ER-α36-mediated nongenomic estrogen signaling increases cisplatin sensitivity in breast cancer cells. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the levels of ER-α36, total EGFR (EGFR) and phosphorylated EGFR (P-EGFR), total HER-2 (HER-2) and phosphorylated HER-2 (P-HER-2), total ERK (ERK) and phosphorylated ERK (P-ERK) were evaluated using western blot. b MCF-7 cells were treated as in ( a ), and then the cell proliferation was measured with CCK-8 assay kit. c MCF-7/ER-α36 cells were treated with 5 μg/mL cisplatin for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the total ERK (ERK) and phosphorylated ERK (P-ERK) was detected by western blot. d MCF-7/ER-α36 cells were treated as in ( c ), and then the cell proliferation was examined using CCK-8 assay kit. * P
Figure Legend Snippet: Disruption of ER-α36-mediated nongenomic estrogen signaling increases cisplatin sensitivity in breast cancer cells. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the levels of ER-α36, total EGFR (EGFR) and phosphorylated EGFR (P-EGFR), total HER-2 (HER-2) and phosphorylated HER-2 (P-HER-2), total ERK (ERK) and phosphorylated ERK (P-ERK) were evaluated using western blot. b MCF-7 cells were treated as in ( a ), and then the cell proliferation was measured with CCK-8 assay kit. c MCF-7/ER-α36 cells were treated with 5 μg/mL cisplatin for 48 h after preincubated with or without AG1478, Lapatinib, and U0126 at the indicated concentrations for 6 h, respectively. Then the total ERK (ERK) and phosphorylated ERK (P-ERK) was detected by western blot. d MCF-7/ER-α36 cells were treated as in ( c ), and then the cell proliferation was examined using CCK-8 assay kit. * P

Techniques Used: Western Blot, CCK-8 Assay

Up-regulation of ER-α36 leads to increased activation of nongenomic estrogen signaling. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h. Then the protein levels of EGFR, HER-2, ER-α36, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. b , c The quantitative analysis of cisplatin-induced expression of ER-α36, EGFR, HER-2 and P-ERK/ERK of ( a ). d MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of EGFR, HER-2, ER-α36, ERK and P-ERK were analyzed by western blot. e The quantitative analysis of cisplatin-induced expression of P-ERK/ERK of ( d ). f MCF-7 cells were treated as in ( a ). The cell lysates were immunoprecipitated with anti-HER-2 or anti-EGFR antibodies. Then the immunoprecipitates were separated by SDS-PAGE and probed with anti-ER-α36 antibodies. Immunoprecipitation of IgG was used as a negative control
Figure Legend Snippet: Up-regulation of ER-α36 leads to increased activation of nongenomic estrogen signaling. a MCF-7 cells were treated with or without 5 μg/mL cisplatin (DDP) for 48 h. Then the protein levels of EGFR, HER-2, ER-α36, total ERK (ERK) and phosphorylated ERK (P-ERK) were detected using western blot. b , c The quantitative analysis of cisplatin-induced expression of ER-α36, EGFR, HER-2 and P-ERK/ERK of ( a ). d MCF-7, BT474 and MDA-MB-231 cells were treated with cisplatin at the indicated concentrations for 48 h and then the protein levels of EGFR, HER-2, ER-α36, ERK and P-ERK were analyzed by western blot. e The quantitative analysis of cisplatin-induced expression of P-ERK/ERK of ( d ). f MCF-7 cells were treated as in ( a ). The cell lysates were immunoprecipitated with anti-HER-2 or anti-EGFR antibodies. Then the immunoprecipitates were separated by SDS-PAGE and probed with anti-ER-α36 antibodies. Immunoprecipitation of IgG was used as a negative control

Techniques Used: Activation Assay, Western Blot, Expressing, Multiple Displacement Amplification, Immunoprecipitation, SDS Page, Negative Control

Related Articles

Western Blot:

Article Title: JAK2 inhibition sensitizes resistant EGFR-mutant lung adenocarcinoma to tyrosine kinase inhibitors
Article Snippet: .. Antibodies used for Western blotting were as follows: anti-pSTAT3 (Y705), total STAT3, pERK (p44/42, T202/Y204), pAKT (S473), total AKT, pS6 (S240/244), S6, pEGFR (Y1068), total EGFR, L858R-EGFR– or del19-EGFR–specific antibodies, anti-JAK2 (Cell Signaling Technology), and anti-JAK1 (BD Biosciences); anti–total ERK, EGFR, Myc, ubiquitin and horseradish peroxidase (HRP)–conjugated anti-rabbit IgG, HRP-conjugated anti-mouse IgG, HRP-conjugated anti-rabbit IgG, SOCS4, and SOCS5 (Santa Cruz Biotechnology); anti–c-MET (Invitrogen); and anti–α-tubulin (Sigma-Aldrich). ..

Article Title: Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis
Article Snippet: .. For phosphorylated EGFR, total EGFR, phosphorylated ERK and total ERK Western blotting, rabbit monoclonal phospho-EGFR (Tyr1068) (1:1000), EGFR (1:1000), phospho-p44/42 ERK1/2 (Thr202/Tyr204) (1:2000) and ERK1/2 antibody (1:1000) from Cell Signaling Technology were used. .. Histology, immunohistochemistry, PAS staining We stained 2 μm sections with hematoxylin and eosin (H & E) or rabbit-anti-human intelectin (94–145) antibody (1:400; Phoenix Pharmaceuticals, US) or an isotype matched control rabbit IgG (1:2000; Abmart Shanghai, China) for endobronchial and skin biopsies.

Incubation:

Article Title: Enhanced cohesion promotes chromosome stability and limits acquired drug resistance in non small cell lung cancer
Article Snippet: .. Antibodies were diluted in 1xTBST/5% milk (phospho EGFR (Cell Signaling), total EGFR (Cell Signaling), total MET (Cell Signaling), total ERK (Cell Signaling), WAPL (Bethyl), Aurora B (BD Biosciences), Histone H3 (Abcam), and alpha-tubulin (Santa Cruz)) or 1xTBST/5%BSA (phospho MET (Cell Signaling), phospho ERK (Cell Signaling)) and incubated at 4° overnight. .. Membranes were washed in 1xTBST and incubated 1h in corresponding secondary antibody (rabbit, mouse: GE Healthcare), washed in 1xTBST buffer and developed using ProSignal Pico (Prometheus).

Polyacrylamide Gel Electrophoresis:

Article Title: Fibrinogen inhibits neurite outgrowth via ?3 integrin-mediated phosphorylation of the EGF receptor
Article Snippet: .. Supernatants were electrophoresed on SDS/4–12% PAGE gels and probed with the following antibodies: phospho-EGFR (Tyr-1173), total-EGFR, and integrin β3 antibodies (1:1,000; Cell Signaling). .. For the inhibition of β3 integrin, neurons were pretreated with a mouse monoclonal anti-rat β3 neutralizing antibody (10 μg/ml; BD Biosciences) or mouse IgG (10 μg/ml; BD Biosciences) for 1 h before the addition of 1.5 mg/ml fibrinogen.

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    Cell Signaling Technology Inc total egfr
    Mutations at the site of drug binding cause resistance in drug sensitive <t>EGFR</t> or <t>HER2</t> exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.
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    Mutations at the site of drug binding cause resistance in drug sensitive EGFR or HER2 exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

    Journal: Cancer research

    Article Title: Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR and HER2 inhibitors

    doi: 10.1158/0008-5472.CAN-16-3404

    Figure Lengend Snippet: Mutations at the site of drug binding cause resistance in drug sensitive EGFR or HER2 exon 20 insertion mutant models. A. EGFR InsGY Ba/F3 cells harboring either a concurrent T790M or a C797S are resistant to dacomitinib or afatinib. Cells were treated with dacomitinib at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. HER2 InsYVMA or InsWLV Ba/F3 cells harboring a concomitant C805S mutation are resistant to neratinib, afatinib and dacomitinib. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing the respective constructs from A. were treated with different drugs at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

    Article Snippet: Anti-phospho-Akt (Ser473), total AKT, phospho EGFR (Tyr1068), total EGFR, phospho Erk (Thr202/Tyr204), total Erk, phospho HER2 (Tyr1221/1222), total HER2, and PARP antibodies were obtained from Cell Signaling Technology.

    Techniques: Binding Assay, Mutagenesis, Expressing, Construct

    EGFR and ERBB2 exon 20 insertions and sensitivity to covalent quinazoline based EGFR inhibitors. A. Ba/F3 cells expressing wild type EGFR or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing different EGFR exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. Ba/F3 cells expressing wild type HER2 or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing different HER2 exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

    Journal: Cancer research

    Article Title: Response heterogeneity of EGFR and HER2 exon 20 insertions to covalent EGFR and HER2 inhibitors

    doi: 10.1158/0008-5472.CAN-16-3404

    Figure Lengend Snippet: EGFR and ERBB2 exon 20 insertions and sensitivity to covalent quinazoline based EGFR inhibitors. A. Ba/F3 cells expressing wild type EGFR or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. B. Ba/F3 cells expressing different EGFR exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins. C. Ba/F3 cells expressing wild type HER2 or different exon 20 insertion mutations. Cells were treated with different drugs at the indicated concentrations, and viable cells were measured after 72 hours of treatment and plotted relative to untreated controls. D. Ba/F3 cells expressing different HER2 exon 20 mutants were treated with gefitinib or dacomitinib at indicated concentrations for 6 hours. Cell extracts were immunoblotted to detect the indicated proteins.

    Article Snippet: Anti-phospho-Akt (Ser473), total AKT, phospho EGFR (Tyr1068), total EGFR, phospho Erk (Thr202/Tyr204), total Erk, phospho HER2 (Tyr1221/1222), total HER2, and PARP antibodies were obtained from Cell Signaling Technology.

    Techniques: Expressing

    Suppression of CIN influences mechanisms of drug tolerance. A) Quantification of anaphase cells exhibiting lagging chromosomes in clonal populations of drug tolerant cells with (Wapl KD) or without (Mock) Wapl depletion. B C) Gefitinib-tolerant PC9 cell clones derived with or without Wapl depletion labelled with centromere enumeration FISH probes for chromosomes 6, 7, 8, and 10 and quantified for intratumor numerical heterogeneity. D) Western blot showing Wapl levels and EGFR pathway status in four each of mock and Wapl-depleted drug tolerant clones. E) qPCR analysis of mRNA levels indicative of EMT pathway activation (slug, vimentin) and MET expression in mock and Wapl-depleted drug tolerant clones.

    Journal: bioRxiv

    Article Title: Enhanced cohesion promotes chromosome stability and limits acquired drug resistance in non small cell lung cancer

    doi: 10.1101/2020.07.10.197350

    Figure Lengend Snippet: Suppression of CIN influences mechanisms of drug tolerance. A) Quantification of anaphase cells exhibiting lagging chromosomes in clonal populations of drug tolerant cells with (Wapl KD) or without (Mock) Wapl depletion. B C) Gefitinib-tolerant PC9 cell clones derived with or without Wapl depletion labelled with centromere enumeration FISH probes for chromosomes 6, 7, 8, and 10 and quantified for intratumor numerical heterogeneity. D) Western blot showing Wapl levels and EGFR pathway status in four each of mock and Wapl-depleted drug tolerant clones. E) qPCR analysis of mRNA levels indicative of EMT pathway activation (slug, vimentin) and MET expression in mock and Wapl-depleted drug tolerant clones.

    Article Snippet: Antibodies were diluted in 1xTBST/5% milk (phospho EGFR (Cell Signaling), total EGFR (Cell Signaling), total MET (Cell Signaling), total ERK (Cell Signaling), WAPL (Bethyl), Aurora B (BD Biosciences), Histone H3 (Abcam), and alpha-tubulin (Santa Cruz)) or 1xTBST/5%BSA (phospho MET (Cell Signaling), phospho ERK (Cell Signaling)) and incubated at 4° overnight.

    Techniques: Clone Assay, Derivative Assay, Fluorescence In Situ Hybridization, Western Blot, Real-time Polymerase Chain Reaction, Activation Assay, Expressing

    DCA activation of ERK1/2 requires EGFR but does not result in phosphorylation of the receptor at Tyr1068

    Journal: Biochimica et biophysica acta

    Article Title: Deoxycholic acid mediates non-canonical EGFR-MAPK activation through the induction of calcium signaling in colon cancer cells

    doi: 10.1016/j.bbalip.2016.04.006

    Figure Lengend Snippet: DCA activation of ERK1/2 requires EGFR but does not result in phosphorylation of the receptor at Tyr1068

    Article Snippet: Primary antibodies that were used in this study include: p-ERK1/2 (Cell Signaling Technologies, Danvers, Ma), total ERK1/2 (Cell Signaling Technologies), p-EGFR Tyr1068 (Cell Signaling Technologies), total EGFR (Cell Signaling Technologies), p-EGFR Tyr845 (Cell Signaling Technologies), p-CAMKII (Santa Cruz Biotechnologies), β-actin (Sigma-Aldrich) and p-c-Src (Cell Signaling Technologies).

    Techniques: Activation Assay

    ITLN1 contributes to HDM-induced IL-25, IL-33 and TSLP expression and the activation of EGFR and ERK pathway in human bronchial epithelial cells. ( a – d ) The kinetics of ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells after exposure to HDM were determined by quantitative PCR. n = 4 wells per group. Data are mean ± SEM. **, P

    Journal: Mucosal immunology

    Article Title: Intelectin contributes to allergen-induced IL-25, IL-33 and TSLP expression and type 2 response in asthma and atopic dermatitis

    doi: 10.1038/mi.2017.10

    Figure Lengend Snippet: ITLN1 contributes to HDM-induced IL-25, IL-33 and TSLP expression and the activation of EGFR and ERK pathway in human bronchial epithelial cells. ( a – d ) The kinetics of ITLN1, IL-25, IL-33 and TSLP expression in BEAS-2B cells after exposure to HDM were determined by quantitative PCR. n = 4 wells per group. Data are mean ± SEM. **, P

    Article Snippet: For phosphorylated EGFR, total EGFR, phosphorylated ERK and total ERK Western blotting, rabbit monoclonal phospho-EGFR (Tyr1068) (1:1000), EGFR (1:1000), phospho-p44/42 ERK1/2 (Thr202/Tyr204) (1:2000) and ERK1/2 antibody (1:1000) from Cell Signaling Technology were used.

    Techniques: Expressing, Activation Assay, Real-time Polymerase Chain Reaction