Structured Review

Cell Signaling Technology Inc egfr
The inhibition of ERK1/2 phosphorylation and function by <t>EGFR</t> and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and <t>RSK1/2/3</t> as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P
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1) Product Images from "Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2"

Article Title: Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

Journal: Carcinogenesis

doi: 10.1093/carcin/bgt257

The inhibition of ERK1/2 phosphorylation and function by EGFR and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and RSK1/2/3 as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P
Figure Legend Snippet: The inhibition of ERK1/2 phosphorylation and function by EGFR and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and RSK1/2/3 as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P

Techniques Used: Inhibition, Western Blot

2) Product Images from "Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation"

Article Title: Ligand-Independent EGFR Activation by Anchorage-Stimulated Src Promotes Cancer Cell Proliferation and Cetuximab Resistance via ErbB3 Phosphorylation

Journal: Cancers

doi: 10.3390/cancers11101552

EGFR ligands are not involved in the phosphorylation of ErbB3. Cells were treated with ( A ) epidermal growth factor (EGF), ( B ) transforming growth factor- α (TGF- α ), ( C ) heparin-binding EGF-like growth factor (HB-EGF), and the phosphorylation levels of EGFR and ErbB3 were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p
Figure Legend Snippet: EGFR ligands are not involved in the phosphorylation of ErbB3. Cells were treated with ( A ) epidermal growth factor (EGF), ( B ) transforming growth factor- α (TGF- α ), ( C ) heparin-binding EGF-like growth factor (HB-EGF), and the phosphorylation levels of EGFR and ErbB3 were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p

Techniques Used: Binding Assay, Western Blot

The phosphorylation levels of focal adhesion kinase (FAK), EGFR, and ErbB3 were reduced in suspension cultures. HSC3, HSC4, SAS, KB, and Ca9-22 cells were cultured on ultra-low adhesion plates (suspension; SS) or tissue culture plates (monolayer; ML). The levels of phosphorylated EGFR, ErbB3, FAK, and integrin β1 were determined by western blotting analysis. α-tubulin was used as the loading control. * Significantly different from control, p
Figure Legend Snippet: The phosphorylation levels of focal adhesion kinase (FAK), EGFR, and ErbB3 were reduced in suspension cultures. HSC3, HSC4, SAS, KB, and Ca9-22 cells were cultured on ultra-low adhesion plates (suspension; SS) or tissue culture plates (monolayer; ML). The levels of phosphorylated EGFR, ErbB3, FAK, and integrin β1 were determined by western blotting analysis. α-tubulin was used as the loading control. * Significantly different from control, p

Techniques Used: Cell Culture, Western Blot

Extracellular signals are not involved in the phosphorylation of ErbB3. Cells were treated with ( A ) neuregulin 1 (NRG1), ( B ) 10% fetal bovine serum (FBS), and ( C ) 10 μM tumor necrosis factor alpha protease inhibitor 2 (TAPI-2) and the phosphorylation levels of EGFR and ErbB3 were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p
Figure Legend Snippet: Extracellular signals are not involved in the phosphorylation of ErbB3. Cells were treated with ( A ) neuregulin 1 (NRG1), ( B ) 10% fetal bovine serum (FBS), and ( C ) 10 μM tumor necrosis factor alpha protease inhibitor 2 (TAPI-2) and the phosphorylation levels of EGFR and ErbB3 were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p

Techniques Used: Protease Inhibitor, Western Blot

Src inhibitor reduces the phosphorylation levels of EGFR, ErbB3, and AKT. HSC3, HSC4, SAS, KB and Ca9-22 cells were treated ( A ) with or without PF573228 or ( B ) Src inhibitor-1. The levels of phosphorylated FAK (pFAK), FAK, phosphorylated EGFR (pEGFR), EGFR, phosphorylated ErbB3 (pErbB3), ErbB3, phosphorylated AKT (pAKT), and AKT were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p
Figure Legend Snippet: Src inhibitor reduces the phosphorylation levels of EGFR, ErbB3, and AKT. HSC3, HSC4, SAS, KB and Ca9-22 cells were treated ( A ) with or without PF573228 or ( B ) Src inhibitor-1. The levels of phosphorylated FAK (pFAK), FAK, phosphorylated EGFR (pEGFR), EGFR, phosphorylated ErbB3 (pErbB3), ErbB3, phosphorylated AKT (pAKT), and AKT were determined by western blotting analysis. α -tubulin was used as the loading control. * Significantly different from control, p

Techniques Used: Western Blot

Models of EGFR-ligand dependent pathway and EGFR-ligand independent pathway in EIS cells. ( A ) In the EGFR-ligand dependent pathway, proliferation is promoted via ERK/cyclin D1 by EGFR activated by the binding of an EGFR-ligand and EGFR. The activity of FAK or sheddase may be involved in EGFR phosphorylation. ( B ) In the EGFR-ligand independent pathway, EGFR transactivated via Src by cell-substratum adhesion phosphorylates ErbB3 and promotes proliferation via AKT.
Figure Legend Snippet: Models of EGFR-ligand dependent pathway and EGFR-ligand independent pathway in EIS cells. ( A ) In the EGFR-ligand dependent pathway, proliferation is promoted via ERK/cyclin D1 by EGFR activated by the binding of an EGFR-ligand and EGFR. The activity of FAK or sheddase may be involved in EGFR phosphorylation. ( B ) In the EGFR-ligand independent pathway, EGFR transactivated via Src by cell-substratum adhesion phosphorylates ErbB3 and promotes proliferation via AKT.

Techniques Used: Impedance Spectroscopy, Binding Assay, Activity Assay

3) Product Images from "Hypoxia‐inducible transgelin 2 selects epithelial‐to‐mesenchymal transition and γ‐radiation‐resistant subtypes by focal adhesion kinase‐associated insulin‐like growth factor 1 receptor activation in non‐small‐cell lung cancer cells. Hypoxia‐inducible transgelin 2 selects epithelial‐to‐mesenchymal transition and γ‐radiation‐resistant subtypes by focal adhesion kinase‐associated insulin‐like growth factor 1 receptor activation in non‐small‐cell lung cancer cells"

Article Title: Hypoxia‐inducible transgelin 2 selects epithelial‐to‐mesenchymal transition and γ‐radiation‐resistant subtypes by focal adhesion kinase‐associated insulin‐like growth factor 1 receptor activation in non‐small‐cell lung cancer cells. Hypoxia‐inducible transgelin 2 selects epithelial‐to‐mesenchymal transition and γ‐radiation‐resistant subtypes by focal adhesion kinase‐associated insulin‐like growth factor 1 receptor activation in non‐small‐cell lung cancer cells

Journal: Cancer Science

doi: 10.1111/cas.13791

Transgelin 2 ( TAGLN 2)‐induced activation of insulin growth factor 1 receptor ( IGF 1R)β/P13K/ AKT pathway stabilizes Snail1 protein by glycogen synthase kinase 3β ( GSK 3β) inactivation: phosphatase and tensin homolog ( PTEN ) independent AKT activation. A, Western blot analysis of cellular p‐ AKT , PI 3K‐P110, p‐ IGF 1Rβ, and p‐epidermal growth factor receptor ( EGFR ) levels in TAGLN 2 ‐overexpressing or TAGLN 2 ‐suppressing A549 and H460 cells. B, Interactions between TAGLN 2, IGF 1Rβ and focal adhesion kinase ( FAK ) were determined using immunoprecipitation assay in TAGLN 2 ‐overexpressing A549 or H460 cells. C, FAK inactivation inhibited the phosphorylation of IGF 1Rβ in TAGLN 2 ‐overexpressing A549 cells (left). Inactivation of IGF 1Rβ also inhibited the phosphorylation of FAK (right). A549 cells were treated with 5 μmol/L FAK inhibitor 14 for 24 h or with 5 μmol/L AG 1024 ( IGF 1R inhibitor) for 12 h. D, Western blot analysis of cellular p‐ FAK and FAK in TAGLN 2 ‐suppressing A549 cells. E, Western blot analysis of IGF 1R signaling pathway and Snail1 in TAGLN 2‐overexpressing non‐small‐cell lung cancer ( NSCLC ) cells by treatment of kinase inhibitors ( AG 1024, LY 294002 : PI 3K inhibitor). F, Western blot analysis of cellular p‐ AKT , p‐ GSK 3β, Snail1, and E‐cadherin levels in MK 2206 ( AKT inhibitor)‐treated TAGLN 2‐ overexpressing NSCLC cells. Tg2, TAGLN2
Figure Legend Snippet: Transgelin 2 ( TAGLN 2)‐induced activation of insulin growth factor 1 receptor ( IGF 1R)β/P13K/ AKT pathway stabilizes Snail1 protein by glycogen synthase kinase 3β ( GSK 3β) inactivation: phosphatase and tensin homolog ( PTEN ) independent AKT activation. A, Western blot analysis of cellular p‐ AKT , PI 3K‐P110, p‐ IGF 1Rβ, and p‐epidermal growth factor receptor ( EGFR ) levels in TAGLN 2 ‐overexpressing or TAGLN 2 ‐suppressing A549 and H460 cells. B, Interactions between TAGLN 2, IGF 1Rβ and focal adhesion kinase ( FAK ) were determined using immunoprecipitation assay in TAGLN 2 ‐overexpressing A549 or H460 cells. C, FAK inactivation inhibited the phosphorylation of IGF 1Rβ in TAGLN 2 ‐overexpressing A549 cells (left). Inactivation of IGF 1Rβ also inhibited the phosphorylation of FAK (right). A549 cells were treated with 5 μmol/L FAK inhibitor 14 for 24 h or with 5 μmol/L AG 1024 ( IGF 1R inhibitor) for 12 h. D, Western blot analysis of cellular p‐ FAK and FAK in TAGLN 2 ‐suppressing A549 cells. E, Western blot analysis of IGF 1R signaling pathway and Snail1 in TAGLN 2‐overexpressing non‐small‐cell lung cancer ( NSCLC ) cells by treatment of kinase inhibitors ( AG 1024, LY 294002 : PI 3K inhibitor). F, Western blot analysis of cellular p‐ AKT , p‐ GSK 3β, Snail1, and E‐cadherin levels in MK 2206 ( AKT inhibitor)‐treated TAGLN 2‐ overexpressing NSCLC cells. Tg2, TAGLN2

Techniques Used: Activation Assay, Western Blot, Immunoprecipitation

4) Product Images from "Pingyangmycin and Bleomycin Share the Same Cytotoxicity Pathway"

Article Title: Pingyangmycin and Bleomycin Share the Same Cytotoxicity Pathway

Journal: Molecules

doi: 10.3390/molecules21070862

Effects of BLM and A5 on epidermal growth factor receptor (EGFR) and phosphorylated-EGFR in A549 and HCT116 cells. Cells were seeded in 10-cm dishes for 24 h followed by treating with serial concentrations of BLM and A5 (0, 10, 20, 40, 80 μM) for another 24 h. The cells were then harvested for immunoblot analysis as described in the Materials and Methods section. The numbers underneath the blots represent band intensity (normalized to β-Actin, the means of three independent experiments) measured by Image J software. The standard deviations (all within ±15% of the means) were not shown. β-Actin was served as an equal loading control. The experiments were repeated three times.
Figure Legend Snippet: Effects of BLM and A5 on epidermal growth factor receptor (EGFR) and phosphorylated-EGFR in A549 and HCT116 cells. Cells were seeded in 10-cm dishes for 24 h followed by treating with serial concentrations of BLM and A5 (0, 10, 20, 40, 80 μM) for another 24 h. The cells were then harvested for immunoblot analysis as described in the Materials and Methods section. The numbers underneath the blots represent band intensity (normalized to β-Actin, the means of three independent experiments) measured by Image J software. The standard deviations (all within ±15% of the means) were not shown. β-Actin was served as an equal loading control. The experiments were repeated three times.

Techniques Used: Software

5) Product Images from "Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy"

Article Title: Genetically distinct glioma stem-like cell xenografts established from paired glioblastoma samples harvested before and after molecularly targeted therapy

Journal: Scientific Reports

doi: 10.1038/s41598-018-37437-2

Pre-EGFR inhibitor GSCs overexpress EGFR and phospho-EGFR and are sensitive to EGFR inhibition. ( A ) RT-PCR showing EGFR expression in the paired GSCs, MGG70R-GSC (70R-GSC) and MGG70RR-GSC (70RR-GSC). GAPDH was used as control. ( B ) Western blotting showing EGFR, phospho-EGFR (Tyr1068), phospho-Akt (Thr308), phospho-Akt (Ser473), Akt, and phospho-Erk1/2 in 70R and 70RR GSCs. Actin served as control. ( C ) GSC sensitivities to molecular targeted agents. Cell viability assay was performed on the paired GSCs after 3-day exposure to the EGFR inhibitor lapatinib (upper), the PI3K inhibitor PF-05212384 (middle) and the MEK inhibitor PD98509 (bottom).
Figure Legend Snippet: Pre-EGFR inhibitor GSCs overexpress EGFR and phospho-EGFR and are sensitive to EGFR inhibition. ( A ) RT-PCR showing EGFR expression in the paired GSCs, MGG70R-GSC (70R-GSC) and MGG70RR-GSC (70RR-GSC). GAPDH was used as control. ( B ) Western blotting showing EGFR, phospho-EGFR (Tyr1068), phospho-Akt (Thr308), phospho-Akt (Ser473), Akt, and phospho-Erk1/2 in 70R and 70RR GSCs. Actin served as control. ( C ) GSC sensitivities to molecular targeted agents. Cell viability assay was performed on the paired GSCs after 3-day exposure to the EGFR inhibitor lapatinib (upper), the PI3K inhibitor PF-05212384 (middle) and the MEK inhibitor PD98509 (bottom).

Techniques Used: Inhibition, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Viability Assay

6) Product Images from "Exposure of Barrett’s and esophageal adenocarcinoma cells to Bile acids activates EGFR-STAT3 signaling axis via induction of APE1"

Article Title: Exposure of Barrett’s and esophageal adenocarcinoma cells to Bile acids activates EGFR-STAT3 signaling axis via induction of APE1

Journal: Oncogene

doi: 10.1038/s41388-018-0388-8

APE1 mediates bile salts-induced STAT3 activation via an EGFR-dependent mechanism Immunoblot analysis of CPB ( A ), FLO-1 ( B ) and OE33 ( C ) cells pretreated with EGFR inhibitor (Gefitinib, 25 μM) followed by exposure to acidic (pH4) bile salts (100 μM). The samples were analyzed for the indicated proteins, β-actin was used as an internal control. ( D ) Immunoblot analysis of OE33 cells with EGFR-knockdown via EGFR siRNA followed by treatment with acidic (pH4) bile salts (100 μM) for 20 minutes and allowed to recover in complete media. The samples were collected at 3 and 6h post recovery and analyzed for the indicated proteins, β-actin was used as an internal control. Immunoprecipitation (IP) of APE1 ( E ), EGFR ( F ) and STAT3 ( G ) in OE33 cells treated with acidic (pH4) bile salts (100 μM) and immunoblotted for the indicated proteins. Results shown are representative of at least three independent experiments.
Figure Legend Snippet: APE1 mediates bile salts-induced STAT3 activation via an EGFR-dependent mechanism Immunoblot analysis of CPB ( A ), FLO-1 ( B ) and OE33 ( C ) cells pretreated with EGFR inhibitor (Gefitinib, 25 μM) followed by exposure to acidic (pH4) bile salts (100 μM). The samples were analyzed for the indicated proteins, β-actin was used as an internal control. ( D ) Immunoblot analysis of OE33 cells with EGFR-knockdown via EGFR siRNA followed by treatment with acidic (pH4) bile salts (100 μM) for 20 minutes and allowed to recover in complete media. The samples were collected at 3 and 6h post recovery and analyzed for the indicated proteins, β-actin was used as an internal control. Immunoprecipitation (IP) of APE1 ( E ), EGFR ( F ) and STAT3 ( G ) in OE33 cells treated with acidic (pH4) bile salts (100 μM) and immunoblotted for the indicated proteins. Results shown are representative of at least three independent experiments.

Techniques Used: Activation Assay, Immunoprecipitation

APE1 co-localizes with EGFR and STAT3 (A and B) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3 with APE1. (C) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3. Protein interactions (red fluorescent signals) were revealed by PLA anti-rabbit plus probe and PLA anti-mouse minus probe in OE33 cells treated with acidic (pH4) bile salts (100 μM) as mentioned in the materials and methods. Nuclei were stained with DAPI (blue). Results shown are representative of at least three independent experiments.
Figure Legend Snippet: APE1 co-localizes with EGFR and STAT3 (A and B) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3 with APE1. (C) In situ proximity ligation assay (PLA) demonstrates the interaction of p-EGFR and p-STAT3. Protein interactions (red fluorescent signals) were revealed by PLA anti-rabbit plus probe and PLA anti-mouse minus probe in OE33 cells treated with acidic (pH4) bile salts (100 μM) as mentioned in the materials and methods. Nuclei were stained with DAPI (blue). Results shown are representative of at least three independent experiments.

Techniques Used: In Situ, Proximity Ligation Assay, Staining

7) Product Images from "Epithelial to Mesenchymal transition, eIF2α phosphorylation and Hsp70 expression enable greater tolerance in A549 cells to TiO2 over ZnO nanoparticles"

Article Title: Epithelial to Mesenchymal transition, eIF2α phosphorylation and Hsp70 expression enable greater tolerance in A549 cells to TiO2 over ZnO nanoparticles

Journal: Scientific Reports

doi: 10.1038/s41598-018-36716-2

( A ) Evaluation of EMT by mRNA level expression of E Cadherin, N Cadherin, EGFR and Clathrin. 24 Hours expression pattern of E Cadherin, N Cadherin, EGFR and Clathrin at the mRNA level was documented in a dose dependent manner. Doses; A- 0, B- 0.15, C-0.31, D-0.62 and E-1.24 mM. The best gel of 3 independent experiments is shown here. Results of the independent experiments were following a similar trend in expression. ( B ) Statistical Analysis mRNA level expression of E Cadherin, N Cadherin, EGFR and Clathrin. Dose dependent expression profile is plotted from the best representative gel for E Cadherin, N Cadherin, EGFR and Clathrin, relative to the internal control GAPDH in response to MeOx NP treatment. P values for ZnO treatment are; E Cadherin- 0.000001773 (***), N Cadherin- 2.75E-06 (***), EGFR- 0.000404547 (***) and Clathrin- 9.05E-06 (***). TiO 2 exposure resulted in P values of; E Cadherin- 0.0001219 (***), N Cadherin- 3.816E-05 (***), EGFR- 4.1E-05 (***) and Clathrin- 0.010 (**). ( C ) Evaluation of EMT through protein level expression of E Cadherin, N Cadherin and EGFR by western blot analysis. Dose dependent expression of E Cadherin, N Cadherin and EGFR is documented to MeOx NP treatment. The best blot of 3 independent experiments is shown here. Results of the independent experiments were following a similar trend in expression. ( D ) Statistical Analysis of EMT markers E Cadherin, N Cadherin along with EGFR at protein level. Dose dependent expression of E Cadherin, N Cadherin and EGFR is plotted from the best representative blot, after normalizing with the internal control; Beta Actin in response to MeOx treatment. P Values for ZnO treatment are; E Cadherin- 0.017406 (*), N Cadherin- 0.00404711 (**) and EGFR- 0.0027940 (**). TiO 2 exposure resulted in p values of; E Cadherin- 0.037336 (*), N Cadherin- 0.0350571 (*) and EGFR- 0.04914 (*).
Figure Legend Snippet: ( A ) Evaluation of EMT by mRNA level expression of E Cadherin, N Cadherin, EGFR and Clathrin. 24 Hours expression pattern of E Cadherin, N Cadherin, EGFR and Clathrin at the mRNA level was documented in a dose dependent manner. Doses; A- 0, B- 0.15, C-0.31, D-0.62 and E-1.24 mM. The best gel of 3 independent experiments is shown here. Results of the independent experiments were following a similar trend in expression. ( B ) Statistical Analysis mRNA level expression of E Cadherin, N Cadherin, EGFR and Clathrin. Dose dependent expression profile is plotted from the best representative gel for E Cadherin, N Cadherin, EGFR and Clathrin, relative to the internal control GAPDH in response to MeOx NP treatment. P values for ZnO treatment are; E Cadherin- 0.000001773 (***), N Cadherin- 2.75E-06 (***), EGFR- 0.000404547 (***) and Clathrin- 9.05E-06 (***). TiO 2 exposure resulted in P values of; E Cadherin- 0.0001219 (***), N Cadherin- 3.816E-05 (***), EGFR- 4.1E-05 (***) and Clathrin- 0.010 (**). ( C ) Evaluation of EMT through protein level expression of E Cadherin, N Cadherin and EGFR by western blot analysis. Dose dependent expression of E Cadherin, N Cadherin and EGFR is documented to MeOx NP treatment. The best blot of 3 independent experiments is shown here. Results of the independent experiments were following a similar trend in expression. ( D ) Statistical Analysis of EMT markers E Cadherin, N Cadherin along with EGFR at protein level. Dose dependent expression of E Cadherin, N Cadherin and EGFR is plotted from the best representative blot, after normalizing with the internal control; Beta Actin in response to MeOx treatment. P Values for ZnO treatment are; E Cadherin- 0.017406 (*), N Cadherin- 0.00404711 (**) and EGFR- 0.0027940 (**). TiO 2 exposure resulted in p values of; E Cadherin- 0.037336 (*), N Cadherin- 0.0350571 (*) and EGFR- 0.04914 (*).

Techniques Used: Expressing, Western Blot

8) Product Images from "Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208"

Article Title: Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2013.156

TM208 inhibited the phosphorylation of EGFR and ERK1/2 in vitro and in vivo . (A) TM208 reduced the expression of p-EGFR (left) and p-ERK1/2 (right) in the MDA-MB-231 cell line. (B) TM208 reduced the expression of p-EGFR (left) and p-ERK1/2 (right) in the MCF-7 cell line. (C) Western blot analysis revealed the inhibition of EGFR phosphorylation (left) and ERK1/2 phosphorylation (right) in the MCF-7 xenograft tumors treated with TM208 (50 and 150 mg/kg). The expression levels of EGFR and ERK1/2 were included as loading controls. Each bar corresponds to the mean±SD of three independent experiments ( n= 3). b P
Figure Legend Snippet: TM208 inhibited the phosphorylation of EGFR and ERK1/2 in vitro and in vivo . (A) TM208 reduced the expression of p-EGFR (left) and p-ERK1/2 (right) in the MDA-MB-231 cell line. (B) TM208 reduced the expression of p-EGFR (left) and p-ERK1/2 (right) in the MCF-7 cell line. (C) Western blot analysis revealed the inhibition of EGFR phosphorylation (left) and ERK1/2 phosphorylation (right) in the MCF-7 xenograft tumors treated with TM208 (50 and 150 mg/kg). The expression levels of EGFR and ERK1/2 were included as loading controls. Each bar corresponds to the mean±SD of three independent experiments ( n= 3). b P

Techniques Used: In Vitro, In Vivo, Expressing, Multiple Displacement Amplification, Western Blot, Inhibition

9) Product Images from "The Prostaglandin E2 Receptor, EP2, Stimulates Keratinocyte Proliferation in Mouse Skin by G Protein-dependent and ?-Arrestin1-dependent Signaling Pathways *"

Article Title: The Prostaglandin E2 Receptor, EP2, Stimulates Keratinocyte Proliferation in Mouse Skin by G Protein-dependent and ?-Arrestin1-dependent Signaling Pathways *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.117689

Butaprost induced β-arrestin1-Src complex formation. A , butaprost increased β-arrestin1-Src complex formation. Mice were sacrificed at 2 h after 50 or 100 nmol of butaprost treatment. Skin lysates (200 μg) were immunoprecipitated ( IP ; non-IP , not immunoprecipitated) with a monoclonal anti-Src antibody, and β-arrestin1 and Src were detected by Western blotting. B , butaprost increased the formation of a β-arrestin1-Src complex. Skin lysates (200 μg) were immunoprecipitated with a polyclonal anti-β-arrestin1 antibody and subjected to Western blot analysis using a monoclonal Src antibody. In A and B , nonspecific IgG light chain served as a control for protein loading and membrane transfer. The number above each lane shows the relative intensities of the bands to IgG light chain. C , butaprost induced phosphorylation of Src and EGFR and increased the β-arrestin1-Src complex in WT but not in β-arrestin1 −/− mice. WT and β-arrestin1 −/− mice were treated for the indicated times with 100 nmol of butaprost. Src and EGFR were immunoprecipitated from protein lysates (200 μg) and subjected to Western blot ( WB ) analysis using an anti-p-Tyr or an anti-β-arrestin1 antibody. D , butaprost induced phosphorylation of ERK1/2, Akt, and STAT3 in WT but not in β-arrestin1 −/− mice. WT and β-arrestin1 −/− mice were treated for the indicated times with 100 nmol of butaprost. Skin lysates were immunoblotted with the indicated antibodies. In C and D , the number above each lane shows the mean-fold intensity as determined by densitometry. In A–D , the data presented are representative of two independent experiments. E , β-arrestin1 deficiency suppressed butaprost-induced keratinocyte proliferation. WT and β-arrestin1 −/− mice were pretreated topically with acetone or 100 nmol of butaprost and sacrificed 24 h following butaprost treatment. *, p
Figure Legend Snippet: Butaprost induced β-arrestin1-Src complex formation. A , butaprost increased β-arrestin1-Src complex formation. Mice were sacrificed at 2 h after 50 or 100 nmol of butaprost treatment. Skin lysates (200 μg) were immunoprecipitated ( IP ; non-IP , not immunoprecipitated) with a monoclonal anti-Src antibody, and β-arrestin1 and Src were detected by Western blotting. B , butaprost increased the formation of a β-arrestin1-Src complex. Skin lysates (200 μg) were immunoprecipitated with a polyclonal anti-β-arrestin1 antibody and subjected to Western blot analysis using a monoclonal Src antibody. In A and B , nonspecific IgG light chain served as a control for protein loading and membrane transfer. The number above each lane shows the relative intensities of the bands to IgG light chain. C , butaprost induced phosphorylation of Src and EGFR and increased the β-arrestin1-Src complex in WT but not in β-arrestin1 −/− mice. WT and β-arrestin1 −/− mice were treated for the indicated times with 100 nmol of butaprost. Src and EGFR were immunoprecipitated from protein lysates (200 μg) and subjected to Western blot ( WB ) analysis using an anti-p-Tyr or an anti-β-arrestin1 antibody. D , butaprost induced phosphorylation of ERK1/2, Akt, and STAT3 in WT but not in β-arrestin1 −/− mice. WT and β-arrestin1 −/− mice were treated for the indicated times with 100 nmol of butaprost. Skin lysates were immunoblotted with the indicated antibodies. In C and D , the number above each lane shows the mean-fold intensity as determined by densitometry. In A–D , the data presented are representative of two independent experiments. E , β-arrestin1 deficiency suppressed butaprost-induced keratinocyte proliferation. WT and β-arrestin1 −/− mice were pretreated topically with acetone or 100 nmol of butaprost and sacrificed 24 h following butaprost treatment. *, p

Techniques Used: Mouse Assay, Immunoprecipitation, Western Blot

Proposed EP2-mediated signaling pathways that contributed to keratinocyte replication and mouse skin papilloma formation. EP2 stimulation by butaprost activated PKA and its downstream effectors, p-GSK3β, p-CREB, and p-ERK1/2. Butaprost also activated EGFR and its downstream effectors, p-ERK1/2, p-STAT3, and p-Akt. EGFR activation involves Src activation via an EP2-β-arrestin1-Src complex. Activation of the PKA and EGFR pathways induced cell proliferation, thereby contributing to skin tumor formation.
Figure Legend Snippet: Proposed EP2-mediated signaling pathways that contributed to keratinocyte replication and mouse skin papilloma formation. EP2 stimulation by butaprost activated PKA and its downstream effectors, p-GSK3β, p-CREB, and p-ERK1/2. Butaprost also activated EGFR and its downstream effectors, p-ERK1/2, p-STAT3, and p-Akt. EGFR activation involves Src activation via an EP2-β-arrestin1-Src complex. Activation of the PKA and EGFR pathways induced cell proliferation, thereby contributing to skin tumor formation.

Techniques Used: Activation Assay

Butaprost induced ERK1/2, Akt, and STAT3 phosphorylation. A , butaprost induced dose-dependent phosphorylation of ERK1/2, Akt, and STAT3. Mice were treated with butaprost (50 or 100 nmol) and sacrificed 2 h after treatment. B , butaprost (100 nmol) induced phosphorylation of ERK1/2, Akt, and STAT3 in a time-dependent manner. C , the Src and EGFR inhibitors suppressed butaprost-induced phosphorylation of ERK1/2, Akt, and STAT3. Mice were pretreated with PP2 (100 nmol) or AG1478 (100 nmol) for 30 min and then treated with 100 nmol of butaprost for 2 h. D , the EGFR inhibitor, gefitinib, suppressed butaprost-induced phosphorylation of EGFR, ERK1/2, Akt, and STAT3. Mice were pretreated with gefitinib (67 nmol) for 30 min and then treated with 100 nmol of butaprost for 2 h. EGFR was immunoprecipitated ( IP ) from skin lysates, and p-EGFR was determined by Western blot ( WB ) analysis by using a monoclonal anti-p-Tyr antibody. In A–D , the phosphorylation status was determined by Western blot analysis using antibodies against indicated proteins. The densitometry data are representative of two independent experiments. In A , C , and D , each lane represents an individual mouse. The number above each lane shows the mean -fold intensity from two mice.
Figure Legend Snippet: Butaprost induced ERK1/2, Akt, and STAT3 phosphorylation. A , butaprost induced dose-dependent phosphorylation of ERK1/2, Akt, and STAT3. Mice were treated with butaprost (50 or 100 nmol) and sacrificed 2 h after treatment. B , butaprost (100 nmol) induced phosphorylation of ERK1/2, Akt, and STAT3 in a time-dependent manner. C , the Src and EGFR inhibitors suppressed butaprost-induced phosphorylation of ERK1/2, Akt, and STAT3. Mice were pretreated with PP2 (100 nmol) or AG1478 (100 nmol) for 30 min and then treated with 100 nmol of butaprost for 2 h. D , the EGFR inhibitor, gefitinib, suppressed butaprost-induced phosphorylation of EGFR, ERK1/2, Akt, and STAT3. Mice were pretreated with gefitinib (67 nmol) for 30 min and then treated with 100 nmol of butaprost for 2 h. EGFR was immunoprecipitated ( IP ) from skin lysates, and p-EGFR was determined by Western blot ( WB ) analysis by using a monoclonal anti-p-Tyr antibody. In A–D , the phosphorylation status was determined by Western blot analysis using antibodies against indicated proteins. The densitometry data are representative of two independent experiments. In A , C , and D , each lane represents an individual mouse. The number above each lane shows the mean -fold intensity from two mice.

Techniques Used: Mouse Assay, Immunoprecipitation, Western Blot

10) Product Images from "Saccharomyces boulardii Inhibits EGF Receptor Signaling and Intestinal Tumor Growth in Apcmin Mice"

Article Title: Saccharomyces boulardii Inhibits EGF Receptor Signaling and Intestinal Tumor Growth in Apcmin Mice

Journal: Gastroenterology

doi: 10.1053/j.gastro.2009.05.050

Immunohistochemical staining showing the in vivo effect of Sb on EGFR and Akt phosphorylation and on cell proliferation in the intestinal tumors of Apc Min mice
Figure Legend Snippet: Immunohistochemical staining showing the in vivo effect of Sb on EGFR and Akt phosphorylation and on cell proliferation in the intestinal tumors of Apc Min mice

Techniques Used: Immunohistochemistry, Staining, In Vivo, Mouse Assay

SbS inactivates EGFR MEK ERK signaling in colon cancer cells
Figure Legend Snippet: SbS inactivates EGFR MEK ERK signaling in colon cancer cells

Techniques Used:

11) Product Images from "USP8 is a novel target for overcoming gefitinib-resistance in lung cancer"

Article Title: USP8 is a novel target for overcoming gefitinib-resistance in lung cancer

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

doi: 10.1158/1078-0432.CCR-12-3696

Inhibition of USP8 enhances co-localization between ubiquitin and target receptor tyrosine kinases. H1650 cells were treated with 1 µM USP8i for 90 min and then immunostained to detect (A) EGFR or (B) ErbB2 (green) and ubiquitin (red). The merged
Figure Legend Snippet: Inhibition of USP8 enhances co-localization between ubiquitin and target receptor tyrosine kinases. H1650 cells were treated with 1 µM USP8i for 90 min and then immunostained to detect (A) EGFR or (B) ErbB2 (green) and ubiquitin (red). The merged

Techniques Used: Inhibition

12) Product Images from "Epidermal Growth Factor Receptor Plays a Significant Role in Hepatocyte Growth Factor Mediated Biological Responses in Mammary Epithelial Cells"

Article Title: Epidermal Growth Factor Receptor Plays a Significant Role in Hepatocyte Growth Factor Mediated Biological Responses in Mammary Epithelial Cells

Journal: Cancer biology & therapy

doi:

Gefitinib inhibited HGF activation c-Met and EGFR. (A) PyVmT cells were serum starved for 20 h treated with 20 ng/ml HGF for the indicated time points. Extracts from each time point were prepared and subjected to immunoblot analyses using phosphotyrosine
Figure Legend Snippet: Gefitinib inhibited HGF activation c-Met and EGFR. (A) PyVmT cells were serum starved for 20 h treated with 20 ng/ml HGF for the indicated time points. Extracts from each time point were prepared and subjected to immunoblot analyses using phosphotyrosine

Techniques Used: Activation Assay

13) Product Images from "Both epidermal growth factor and insulin-like growth factor receptors are dispensable for structural intestinal adaptation"

Article Title: Both epidermal growth factor and insulin-like growth factor receptors are dispensable for structural intestinal adaptation

Journal: Journal of pediatric surgery

doi: 10.1016/j.jpedsurg.2015.03.015

Rates of crypt cell proliferation at baseline and 7 days after 50% proximal small bowel resection (SBR) in EGFR/IGF1R-IKO (n=6) and wild-type (WT; n=7) littermates. P-histone 3 immunostaining was performed on tissue sections and positively stained cells were counted in each crypt. A ratio was calculated as number of cells stained positive divided by total number of cells in the crypts. A minimum of 20 crypts were counted per mouse.
Figure Legend Snippet: Rates of crypt cell proliferation at baseline and 7 days after 50% proximal small bowel resection (SBR) in EGFR/IGF1R-IKO (n=6) and wild-type (WT; n=7) littermates. P-histone 3 immunostaining was performed on tissue sections and positively stained cells were counted in each crypt. A ratio was calculated as number of cells stained positive divided by total number of cells in the crypts. A minimum of 20 crypts were counted per mouse.

Techniques Used: Immunostaining, Staining

A) Western blot confirming deletion of EGFR and IGF1R protein expression in crypt enterocytes. Both EGFR and IGF1R expression were knocked out in the intestinal epithelium in adult mice (EGFR/IGF1R-IKO; n=6) following the injection of tamoxifen. Both the knockout mice and wild-type (n=7) controls were administered tamoxifen for three days prior to resection. Tubulin was used as loading control. Successful deletion of EGFR and IGF1R protein was confirmed with all Villin Cre-ER(+); EGFR (f/f), IGF1R (f/f) mice. B ) Percentage increase in crypt depth and villus height for EGFR/IGF1R-IKO (n=6) and WT (n=7) mice after small bowel resection. There were no statistical differences in postoperative villus or crypt growth between groups.
Figure Legend Snippet: A) Western blot confirming deletion of EGFR and IGF1R protein expression in crypt enterocytes. Both EGFR and IGF1R expression were knocked out in the intestinal epithelium in adult mice (EGFR/IGF1R-IKO; n=6) following the injection of tamoxifen. Both the knockout mice and wild-type (n=7) controls were administered tamoxifen for three days prior to resection. Tubulin was used as loading control. Successful deletion of EGFR and IGF1R protein was confirmed with all Villin Cre-ER(+); EGFR (f/f), IGF1R (f/f) mice. B ) Percentage increase in crypt depth and villus height for EGFR/IGF1R-IKO (n=6) and WT (n=7) mice after small bowel resection. There were no statistical differences in postoperative villus or crypt growth between groups.

Techniques Used: Western Blot, Expressing, Mouse Assay, Injection, Knock-Out

Angiogenesis at baseline and 7 days after 50% proximal small bowel resection (SBR) as measured by counting the number of cd31 positive-stained vessels per high power field. Ten high power fields were counted and averaged. Both EGFR/IGF1R-IKO (n=6) and wild-type (WT; n=7) mice showed an increase in cd31 positive stained vessels per high power field after SBR. However, EGFR/IGF1R-IKO mice had an impaired angiogenic response compared with the WT group (p-value
Figure Legend Snippet: Angiogenesis at baseline and 7 days after 50% proximal small bowel resection (SBR) as measured by counting the number of cd31 positive-stained vessels per high power field. Ten high power fields were counted and averaged. Both EGFR/IGF1R-IKO (n=6) and wild-type (WT; n=7) mice showed an increase in cd31 positive stained vessels per high power field after SBR. However, EGFR/IGF1R-IKO mice had an impaired angiogenic response compared with the WT group (p-value

Techniques Used: Staining, Mouse Assay, Significance Assay

Western blot demonstrates phosphorylation of ERK (Thr 202/Tyr 204) and AKT (Ser 473) pathways in both EGFR/IGF1R-IKO and WT mice in crypt enterocytes. Actin was used as a loading control.
Figure Legend Snippet: Western blot demonstrates phosphorylation of ERK (Thr 202/Tyr 204) and AKT (Ser 473) pathways in both EGFR/IGF1R-IKO and WT mice in crypt enterocytes. Actin was used as a loading control.

Techniques Used: Western Blot, Mouse Assay

14) Product Images from "HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold"

Article Title: HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00811-14

HUWE1 regulates the stability of RAF-1. (A) 293FT, Cos1, or HeLa cells were transiently transfected with HUWE1 siRNA. At 48 h after transfection, cells were harvested for immunoblotting. The expression of Shoc2, HUWE1, B-RAF, A-RAF, RAF-1, EGFR, MEK1/2,
Figure Legend Snippet: HUWE1 regulates the stability of RAF-1. (A) 293FT, Cos1, or HeLa cells were transiently transfected with HUWE1 siRNA. At 48 h after transfection, cells were harvested for immunoblotting. The expression of Shoc2, HUWE1, B-RAF, A-RAF, RAF-1, EGFR, MEK1/2,

Techniques Used: Transfection, Expressing

15) Product Images from "Epidermal growth factor receptor restoration rescues the fatty liver regeneration in mice"

Article Title: Epidermal growth factor receptor restoration rescues the fatty liver regeneration in mice

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00032.2017

Reduced EGFR expression with increasing obesity in mice. A : Western blotting shows reduced EGFR and pY1068-EGFR in normal, freely fed lean (10% fat diet) and diet-induced obese (DIO) mice (60% fat diet). Each lane represents an individual mouse. Representative result of duplicate experiments. B : linear regression analysis of EGFR or pY1068-EGFR levels, normalized to GAPDH, vs. percent body fat determined by EchoMRI in the conscious mouse. No significant (N.S.) relationship was seen for lean mice or for DIO mice with EGFR/pEGFR vs. total body weight or epididymal fat pad mass. C : EGFR immunohistochemical staining of human fatty liver, normal human liver, and hepatocellular carcinoma tissue sections shows lowest staining in fatty liver. Note lack of discernible localization of signal to the cell membrane in fatty liver.
Figure Legend Snippet: Reduced EGFR expression with increasing obesity in mice. A : Western blotting shows reduced EGFR and pY1068-EGFR in normal, freely fed lean (10% fat diet) and diet-induced obese (DIO) mice (60% fat diet). Each lane represents an individual mouse. Representative result of duplicate experiments. B : linear regression analysis of EGFR or pY1068-EGFR levels, normalized to GAPDH, vs. percent body fat determined by EchoMRI in the conscious mouse. No significant (N.S.) relationship was seen for lean mice or for DIO mice with EGFR/pEGFR vs. total body weight or epididymal fat pad mass. C : EGFR immunohistochemical staining of human fatty liver, normal human liver, and hepatocellular carcinoma tissue sections shows lowest staining in fatty liver. Note lack of discernible localization of signal to the cell membrane in fatty liver.

Techniques Used: Expressing, Mouse Assay, Western Blot, Immunohistochemistry, Staining

16) Product Images from "Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf"

Article Title: Small-molecule inhibitors of ERK-mediated immediate early gene expression and proliferation of melanoma cells expressing mutated BRaf

Journal: The Biochemical journal

doi: 10.1042/BJ20131571

Selective inhibition of ERK-mediated phosphorylation of substrates by compounds ( A ) Structures of the seven diverse compounds identified by CADD and initially tested. ( B ) HeLa cells were pre-incubated for 30 min with indicated compounds (100 μ M) followed by treatment with EGF (25 ng/ml) for 10 min to activate ERK1/2 signalling. Lysates were immunoblotted for phosphorylated Elk-1 (pElk-1 Ser 383 ) or active ERK1/2 (pERK1/2). ( C ) Following treatment as in (B), immunoblot analysis of lysates from cells treated with 2.3.2 suggested selective phosphorylation inhibition of Elk-1 as compared with RSK1 (pElk-1 Ser 383 and pRSK1 Thr 573 ). Phosphorylation of ERK1/2 and, MEK1/2 (pERK1/2 and pMEK1/2) or Tyr 1068 autophosphorylation of EGFR (pEGFR Tyr 1068 ) was not affected by 2.3.2. The MEK1/2 inhibitor U0126 (10 μ M) was used to inhibit all ERK1/2 signalling, and α -tubulin expression was used as a protein loading control.
Figure Legend Snippet: Selective inhibition of ERK-mediated phosphorylation of substrates by compounds ( A ) Structures of the seven diverse compounds identified by CADD and initially tested. ( B ) HeLa cells were pre-incubated for 30 min with indicated compounds (100 μ M) followed by treatment with EGF (25 ng/ml) for 10 min to activate ERK1/2 signalling. Lysates were immunoblotted for phosphorylated Elk-1 (pElk-1 Ser 383 ) or active ERK1/2 (pERK1/2). ( C ) Following treatment as in (B), immunoblot analysis of lysates from cells treated with 2.3.2 suggested selective phosphorylation inhibition of Elk-1 as compared with RSK1 (pElk-1 Ser 383 and pRSK1 Thr 573 ). Phosphorylation of ERK1/2 and, MEK1/2 (pERK1/2 and pMEK1/2) or Tyr 1068 autophosphorylation of EGFR (pEGFR Tyr 1068 ) was not affected by 2.3.2. The MEK1/2 inhibitor U0126 (10 μ M) was used to inhibit all ERK1/2 signalling, and α -tubulin expression was used as a protein loading control.

Techniques Used: Inhibition, Incubation, Expressing

17) Product Images from "Transforming Growth Factor Alpha (TGF?) Regulates Granulosa Cell Tumor (GCT) Cell Proliferation and Migration through Activation of Multiple Pathways"

Article Title: Transforming Growth Factor Alpha (TGF?) Regulates Granulosa Cell Tumor (GCT) Cell Proliferation and Migration through Activation of Multiple Pathways

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048299

Effect of TGFα and kinase inhibitors on KGN cell cycle progression. KGN cells were treated without (Control) or with TGFα (10 ng/ml) and/or kinase inhibitors for 24 hours and cell cycle progression was determined by flow cytometry. AG1478 (100 nM): EGFR kinase inhibitor; U0126 (4 µM): MEK inhibitor; Rapamycin (20 nM): mTOR inhibitor; LY294002 (100 nM), PI3K inhibitor. Results are representative of three separate experiments.
Figure Legend Snippet: Effect of TGFα and kinase inhibitors on KGN cell cycle progression. KGN cells were treated without (Control) or with TGFα (10 ng/ml) and/or kinase inhibitors for 24 hours and cell cycle progression was determined by flow cytometry. AG1478 (100 nM): EGFR kinase inhibitor; U0126 (4 µM): MEK inhibitor; Rapamycin (20 nM): mTOR inhibitor; LY294002 (100 nM), PI3K inhibitor. Results are representative of three separate experiments.

Techniques Used: Flow Cytometry, Cytometry

18) Product Images from "Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line"

Article Title: Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2013.115

Merged signaling pathways in SNU216-LR. ( a ) Reconstructed EGFR/HER2 and MET signaling pathways with two downstream signaling pathways (PI3K/AKT and MAPK/ERK). Red, blue and yellow closed circles on each protein represent increased, decreased and newly identified phosphorylation sites in SNU216-LR, respectively. Specific phosphorylated sites (marked with an asterisk) of HER2, EGFR, MET, SRC, C-RAF and MAPK were also identified from immunoblot analysis with antibodies against p-EGFR (pY1068), p-HER2 (pY1221/1222), p-MET (pY1234/1235), p-AKT (pS473), p-MAPK (pY202/pY204), p-SRC (pY416) and p-RAF (pS338). Protein names for symbols are as follows: AKT, α-serine/threonine-protein kinase; C-RAF, RAF proto-oncogene serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HER2, human epidermal growth factor receptor 2; MAPK, mitogen-activated protein kinase 1; GRB2, growth factor receptor-bound protein 2; MET, hepatocyte growth factor/mesenchymal–epithelial transition factor; PI3K, phosphatidylinositide 3-kinase; RAC1, Ras-related C3 botulinum toxin substrate 1; RAS, Ras-related protein R-Ras; SOS, son of sevenless homolog; SRC, proto-oncogene tyrosine protein kinase Src. ( b ) Immunoblot analysis of SNU216 and three different resistant clones (SNU216-LR 1, 2 and 3) with phospho-tyrosine or serine-specific antibodies. α-Tubulin was used as a loading control. Data are representative of three independent experiments.
Figure Legend Snippet: Merged signaling pathways in SNU216-LR. ( a ) Reconstructed EGFR/HER2 and MET signaling pathways with two downstream signaling pathways (PI3K/AKT and MAPK/ERK). Red, blue and yellow closed circles on each protein represent increased, decreased and newly identified phosphorylation sites in SNU216-LR, respectively. Specific phosphorylated sites (marked with an asterisk) of HER2, EGFR, MET, SRC, C-RAF and MAPK were also identified from immunoblot analysis with antibodies against p-EGFR (pY1068), p-HER2 (pY1221/1222), p-MET (pY1234/1235), p-AKT (pS473), p-MAPK (pY202/pY204), p-SRC (pY416) and p-RAF (pS338). Protein names for symbols are as follows: AKT, α-serine/threonine-protein kinase; C-RAF, RAF proto-oncogene serine/threonine-protein kinase; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HER2, human epidermal growth factor receptor 2; MAPK, mitogen-activated protein kinase 1; GRB2, growth factor receptor-bound protein 2; MET, hepatocyte growth factor/mesenchymal–epithelial transition factor; PI3K, phosphatidylinositide 3-kinase; RAC1, Ras-related C3 botulinum toxin substrate 1; RAS, Ras-related protein R-Ras; SOS, son of sevenless homolog; SRC, proto-oncogene tyrosine protein kinase Src. ( b ) Immunoblot analysis of SNU216 and three different resistant clones (SNU216-LR 1, 2 and 3) with phospho-tyrosine or serine-specific antibodies. α-Tubulin was used as a loading control. Data are representative of three independent experiments.

Techniques Used: Clone Assay

19) Product Images from "FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy"

Article Title: FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

Journal: Scientific Reports

doi: 10.1038/srep34316

Fak deficiency accelerates proliferation of hepatocytes after PHx by enhancing activation of EGFR. (A) Left, expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. Right, quantification of western blotting by Image J software. (B) Expression of p-MET, MET and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx (n = 6). (E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6). (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6).
Figure Legend Snippet: Fak deficiency accelerates proliferation of hepatocytes after PHx by enhancing activation of EGFR. (A) Left, expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. Right, quantification of western blotting by Image J software. (B) Expression of p-MET, MET and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx. (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or 50 mg/kg erlotinib by oral gavage daily for 3 days beginning one day before PHx (n = 6). (E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6). (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice for (D) (n = 6).

Techniques Used: Activation Assay, Expressing, Mouse Assay, Western Blot, Software, Immunohistochemistry

Fak deficiency increases HB-EGF and proliferation of hepatocytes after PHx by increasing the expression of TNFα. (A) TNFα mRNA (top) and protein (bottom) expression levels in the whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF mRNA expression level in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by i.p. injection daily for 3 days starting one day prior to PHx. (C) expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in the Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification (n = 6) of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) . (E) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 5) for (D) .
Figure Legend Snippet: Fak deficiency increases HB-EGF and proliferation of hepatocytes after PHx by increasing the expression of TNFα. (A) TNFα mRNA (top) and protein (bottom) expression levels in the whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF mRNA expression level in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by i.p. injection daily for 3 days starting one day prior to PHx. (C) expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in the Hep WT and Hep ∆Fak mice treated with either vehicle or a neutralized TNFα antibody by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification (n = 6) of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice for (D) . (E) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 5) for (D) .

Techniques Used: Expressing, Mouse Assay, Injection, Immunohistochemistry

Fak deficiency increases EGFR activation and proliferation of hepatocytes after PHx by increasing expression of HB-EGF. (A) HB-EGF , TGFα , EGF and AREG mRNA expression levels in whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF protein expression levels in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) . (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) .
Figure Legend Snippet: Fak deficiency increases EGFR activation and proliferation of hepatocytes after PHx by increasing expression of HB-EGF. (A) HB-EGF , TGFα , EGF and AREG mRNA expression levels in whole livers of Hep WT and Hep ∆Fak mice 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (B) HB-EGF protein expression levels in whole livers of Hep WT and Hep ∆Fak mice (pooled samples from 3 mice) 0, 1, 1.5, 2, 3, 5 and 7 days after PHx (n = 6). (C) Expression of p-EGFR, EGFR and β-actin proteins in whole livers of Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx. (D) Liver weight/body weight ratios were analyzed in Hep WT and Hep ∆Fak mice treated with either vehicle or CRM197 by oral gavage daily for 3 days starting one day before PHx (n = 6). ( E) Representative photomicrographs and quantification of immunohistochemistry for Ki67 in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) . (F) Representative photomicrographs and quantification of immunohistochemistry for BrdU in the livers of Hep WT and Hep ∆Fak mice (n = 6) for (D) .

Techniques Used: Activation Assay, Expressing, Mouse Assay, Immunohistochemistry

20) Product Images from "BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer"

Article Title: BAG3 promotes tumour cell proliferation by regulating EGFR signal transduction pathways in triple negative breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.24590

Silencing BAG3 reduces activation of the AKT and FAK signalling pathways which regulate proliferation in TNBC cell lines ( A ) MDA-MB-468 cells were treated with siControl or siBAG3 and EGF pathway analysis performed on lysates using an EGF phospho antibody array. Quantitative histograms of PI3K/AKT signalling components from the EGF array are displayed. The histograms represent average protein expression (± SD) ( n = 6). ( B ) The protein expression of pAktSer473, pAktThr308, pmTORSer2441, pIKKα/βSer180/181, Akt, mTOR, IKKα/β and BAG3 was confirmed by immunoblotting. ( C ) Quantitative histograms of FAK/Src pathway components. The histograms represent average protein expression (± SD) ( n = 6). ( D ) The protein expression of pSrcTyr418, Src, pFAKTyr397, pFAKTyr576, pFAKTyr925, FAK, pSTAT1Ser727, STAT1 was confirmed by immunoblotting. ( E ) A hypothetical model of potential regulation of EGFR downstream signaling pathways by BAG3. The diagram was produced using Servier Medical Art ( F ) The protein expression of pFAKTyr397, FAK, pAKTSer473, AKT in BT-549 cells treated with siBAG3 and siControl was confirmed by immunoblotting. ( G ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in MDA-MB-468 and BT-549 cell lines treated with an additional siRNA sequence (S2) targeting BAG3 and was confirmed by immunoblotting. ( H ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in HCC1937 cells treated with FlagBAG3 and siControl was confirmed by immunoblotting. ( I ) BT-549 and MDA-MB-468 cells were treated with 5 uM MK-2206 and FAK14. Reduced activation of pFAKTyr397 after FAK14 treatment was confirmed by immunoblotting. ( J ) Reduced activation of pAKTSer473 after MK-2206 treatment was confirmed by immunoblotting. ( K ) A quantitative graph of proliferation relative to the control in MDA-MB-468, and BT-549 cells after treating with FAK and MK-2206 inhibitors. The histograms represent mean ( ± SD) Brdu incorporation relative to the control ( n = 3). An asterisk represents p
Figure Legend Snippet: Silencing BAG3 reduces activation of the AKT and FAK signalling pathways which regulate proliferation in TNBC cell lines ( A ) MDA-MB-468 cells were treated with siControl or siBAG3 and EGF pathway analysis performed on lysates using an EGF phospho antibody array. Quantitative histograms of PI3K/AKT signalling components from the EGF array are displayed. The histograms represent average protein expression (± SD) ( n = 6). ( B ) The protein expression of pAktSer473, pAktThr308, pmTORSer2441, pIKKα/βSer180/181, Akt, mTOR, IKKα/β and BAG3 was confirmed by immunoblotting. ( C ) Quantitative histograms of FAK/Src pathway components. The histograms represent average protein expression (± SD) ( n = 6). ( D ) The protein expression of pSrcTyr418, Src, pFAKTyr397, pFAKTyr576, pFAKTyr925, FAK, pSTAT1Ser727, STAT1 was confirmed by immunoblotting. ( E ) A hypothetical model of potential regulation of EGFR downstream signaling pathways by BAG3. The diagram was produced using Servier Medical Art ( F ) The protein expression of pFAKTyr397, FAK, pAKTSer473, AKT in BT-549 cells treated with siBAG3 and siControl was confirmed by immunoblotting. ( G ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in MDA-MB-468 and BT-549 cell lines treated with an additional siRNA sequence (S2) targeting BAG3 and was confirmed by immunoblotting. ( H ) The protein expression of pFAKTyr397, pAKTSer473 and GAPDH in HCC1937 cells treated with FlagBAG3 and siControl was confirmed by immunoblotting. ( I ) BT-549 and MDA-MB-468 cells were treated with 5 uM MK-2206 and FAK14. Reduced activation of pFAKTyr397 after FAK14 treatment was confirmed by immunoblotting. ( J ) Reduced activation of pAKTSer473 after MK-2206 treatment was confirmed by immunoblotting. ( K ) A quantitative graph of proliferation relative to the control in MDA-MB-468, and BT-549 cells after treating with FAK and MK-2206 inhibitors. The histograms represent mean ( ± SD) Brdu incorporation relative to the control ( n = 3). An asterisk represents p

Techniques Used: Activation Assay, Multiple Displacement Amplification, Ab Array, Expressing, Produced, Sequencing, BrdU Incorporation Assay

BAG3 interacts with EGFR and components of the EGFR pathway ( A ) Protein expression levels of BAG3, pEGFRTyr1173, EGFR and GAPDH in 8 TNBC cell lines and 1 normal breast epithelial cell line 184B5 analysed by immunoblotting. ( B ) Pearson Correlation Analysis for BAG3 versus EGFR expression in 8 TNBC cell lines is displayed. ( C ) BAG3 was immunoprecipitated (IP) from MDA-MB-468 and BT-549 cells and enrichment of BAG3 by immunoblotting is displayed. The number of BAG3 peptides enriched in the IP identified by mass spectrometry are also displayed (table). ( D) Proteins identified in the BAG3 Interactome (IP) by mass spectrometry were analysed using the PANTHER Classification System. The top 8 Panther Pathways (as determined by gene enrichment) represented in the BAG3 Interactome are listed ( E ) EGF signalling proteins identified in the BAG3 Interactome of BT-549 and MDA-MB-468 cells are listed ( F ) Immunoblotting was performed for BAG3, EGFR, PLCγ, STAT1 and Src in the BAG3 Interactome isolated from BT-549 cells ( G ) Immunoblotting was performed for BAG3, EGFR, PLCγ, STAT1 and Src in the BAG3 Interactome isolated from MDA-MB-468 cells. ( H ) The protein expression of BAG3, EGFR and GAPDH in MDA-MB-468 cells treated with siBAG3 and siControl. ( I ) A quantitative graph of EGFR protein expression in MDA-MB-468 cells treated with siBAG3 and siControl. The histograms represent the average protein expression of EGFR (± SD) ( n = 3).
Figure Legend Snippet: BAG3 interacts with EGFR and components of the EGFR pathway ( A ) Protein expression levels of BAG3, pEGFRTyr1173, EGFR and GAPDH in 8 TNBC cell lines and 1 normal breast epithelial cell line 184B5 analysed by immunoblotting. ( B ) Pearson Correlation Analysis for BAG3 versus EGFR expression in 8 TNBC cell lines is displayed. ( C ) BAG3 was immunoprecipitated (IP) from MDA-MB-468 and BT-549 cells and enrichment of BAG3 by immunoblotting is displayed. The number of BAG3 peptides enriched in the IP identified by mass spectrometry are also displayed (table). ( D) Proteins identified in the BAG3 Interactome (IP) by mass spectrometry were analysed using the PANTHER Classification System. The top 8 Panther Pathways (as determined by gene enrichment) represented in the BAG3 Interactome are listed ( E ) EGF signalling proteins identified in the BAG3 Interactome of BT-549 and MDA-MB-468 cells are listed ( F ) Immunoblotting was performed for BAG3, EGFR, PLCγ, STAT1 and Src in the BAG3 Interactome isolated from BT-549 cells ( G ) Immunoblotting was performed for BAG3, EGFR, PLCγ, STAT1 and Src in the BAG3 Interactome isolated from MDA-MB-468 cells. ( H ) The protein expression of BAG3, EGFR and GAPDH in MDA-MB-468 cells treated with siBAG3 and siControl. ( I ) A quantitative graph of EGFR protein expression in MDA-MB-468 cells treated with siBAG3 and siControl. The histograms represent the average protein expression of EGFR (± SD) ( n = 3).

Techniques Used: Expressing, Immunoprecipitation, Multiple Displacement Amplification, Mass Spectrometry, Isolation

21) Product Images from "Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial–mesenchymal transition is critical in conquering ALK-positive lung cancer"

Article Title: Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial–mesenchymal transition is critical in conquering ALK-positive lung cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.25531

Establishment of ALK-TKI–resistant H2228 cells (A) H2228-CRR, H2228-ALR and H2228-CER cells are resistant to crizotinib, alectinib and ceritinib, respectively. The results of cell viability assays are shown. Data, mean ± SEM from three independent experiments. (B) FISH analysis shows a decrease of the ALK fusion gene in ALK-TKI–resistant H2228 cells compared with H2228 cells (red, ALK3’ ; green, ALK5’ ). The percentage of ALK-FISH positive cells are shown in the lower right corners. (C) Protein levels of p-ALK, ALK, p-EGFR, EGFR, p-AKT, AKT, p-ERK and ERK were analyzed by western blotting. ALK-TKI–resistant cells showed markedly decreased p-ALK and ALK expressions.
Figure Legend Snippet: Establishment of ALK-TKI–resistant H2228 cells (A) H2228-CRR, H2228-ALR and H2228-CER cells are resistant to crizotinib, alectinib and ceritinib, respectively. The results of cell viability assays are shown. Data, mean ± SEM from three independent experiments. (B) FISH analysis shows a decrease of the ALK fusion gene in ALK-TKI–resistant H2228 cells compared with H2228 cells (red, ALK3’ ; green, ALK5’ ). The percentage of ALK-FISH positive cells are shown in the lower right corners. (C) Protein levels of p-ALK, ALK, p-EGFR, EGFR, p-AKT, AKT, p-ERK and ERK were analyzed by western blotting. ALK-TKI–resistant cells showed markedly decreased p-ALK and ALK expressions.

Techniques Used: Fluorescence In Situ Hybridization, Western Blot

22) Product Images from "Development of parallel reaction monitoring (PRM)-based quantitative proteomics applied to HER2-Positive breast cancer"

Article Title: Development of parallel reaction monitoring (PRM)-based quantitative proteomics applied to HER2-Positive breast cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.26031

Protein expression of breast cell lines for EGFR, HER2, HER3 and PTEN and the corresponding proteomic classifications of Ginestier, ICC, HER2 expression obtained using western blot and transcriptomic classification SSPHU HeatMap ( A ) and graphic representations of Principal Component analysis ( B ).
Figure Legend Snippet: Protein expression of breast cell lines for EGFR, HER2, HER3 and PTEN and the corresponding proteomic classifications of Ginestier, ICC, HER2 expression obtained using western blot and transcriptomic classification SSPHU HeatMap ( A ) and graphic representations of Principal Component analysis ( B ).

Techniques Used: Expressing, Immunocytochemistry, Western Blot

23) Product Images from "UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer"

Article Title: UCH-L1-mediated Down-regulation of Estrogen Receptor α Contributes to Insensitivity to Endocrine Therapy for Breast Cancer

Journal: Theranostics

doi: 10.7150/thno.39814

UCH-L1 regulates the transcription of ERα gene via EGFR pathway. (A) MCF-7 or T47D cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid. (B and C) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or UCH-L1 siRNAs for 72h (B) , or were treated with 10 μM LDN for 24h (C) . The ERα mRNA level was analyzed by real-time PCR. (D) MCF-7 cells were transfected with a control plasmid or a Flag-EGFR plasmid. The mRNA level of ERα was measured by real-time PCR. The expressions of EGFR and ERα were measured by western blot. β-actin was used as a loading control. Results shown are Mean ± s.d., n=3. ∗, p
Figure Legend Snippet: UCH-L1 regulates the transcription of ERα gene via EGFR pathway. (A) MCF-7 or T47D cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid. (B and C) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or UCH-L1 siRNAs for 72h (B) , or were treated with 10 μM LDN for 24h (C) . The ERα mRNA level was analyzed by real-time PCR. (D) MCF-7 cells were transfected with a control plasmid or a Flag-EGFR plasmid. The mRNA level of ERα was measured by real-time PCR. The expressions of EGFR and ERα were measured by western blot. β-actin was used as a loading control. Results shown are Mean ± s.d., n=3. ∗, p

Techniques Used: Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

UCH-L1 deubiquitinates and stabilizes EGFR. (A) MCF-7 cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (B) Increasing amounts (0μg, 0.5μg, 1.5μg, 3μg) of UCH-L1 plasmid were transfected into HEK293 cells, and the expression of EGFR was measured by western blot. (C) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or UCH-L1 siRNAs. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (D) MCF-7/AdrR cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, followed by transfection with a siRNA-resistant myc-his-UCH-L1 expression plasmid. The expressions of EGFR and Myc were measured by western blot. β-actin was used as a loading control. (E) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, followed by treatment with 20μM MG132 for 4h. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (F and G) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, and then subjected to cycloheximide (10μg/ml) chase at the indicated time (F) . HEK293T cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid, and then subjected to cycloheximide (10μg/ml) chase at the indicated time (G) . The expression of EGFR was measured by western blot. β-actin was used as a loading control. (H) HEK293T cells were transfected with Flag-EGFR and myc-his-UCH-L1 plasmids, and then subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies. The lysates and immunoprecipitates were then blotted. (I) MCF-7/AdrR cells transfected with myc-his-UCH-L1 plasmid were subjected immunoprecipitation with anti-Myc antibodies. The lysates and immunoprecipitates were analyzed. (J) Endogenous UCH-L1 and EGFR proteins interact with one another in MCF-7/AdrR cells. Endogenous EGFR proteins were immunoprecipitated with the anti-EGFR antibody. Endogenous UCH-L1 was detected by WB. (K) HEK293T cells transfected with Flag-EGFR were lysed and lysates were incubated with GST or GST-UCH-L1-GSH-Sepharose. Proteins retained on Sepharose were blotted with the indicated antibodies. (L and M) HEK293T cells transfected with the indicated constructs were treated with MG132 (20μM) for 8 hours before harvest. EGFR was immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-HA antibodies. (N) Ubiquitinated EGFR was purified from MG132-treated HEK293T cells and then incubated with purified GST or GST-UCH-L1 in vitro, and then blotted with anti-HA antibodies.
Figure Legend Snippet: UCH-L1 deubiquitinates and stabilizes EGFR. (A) MCF-7 cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (B) Increasing amounts (0μg, 0.5μg, 1.5μg, 3μg) of UCH-L1 plasmid were transfected into HEK293 cells, and the expression of EGFR was measured by western blot. (C) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or UCH-L1 siRNAs. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (D) MCF-7/AdrR cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, followed by transfection with a siRNA-resistant myc-his-UCH-L1 expression plasmid. The expressions of EGFR and Myc were measured by western blot. β-actin was used as a loading control. (E) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, followed by treatment with 20μM MG132 for 4h. The expressions of UCH-L1 and EGFR were measured by western blot. β-actin was used as a loading control. (F and G) HCC1806 or BT549 cells were transfected with a non-targeting siRNA or an UCH-L1 siRNA, and then subjected to cycloheximide (10μg/ml) chase at the indicated time (F) . HEK293T cells were transfected with a control plasmid or a myc-his-UCH-L1 plasmid, and then subjected to cycloheximide (10μg/ml) chase at the indicated time (G) . The expression of EGFR was measured by western blot. β-actin was used as a loading control. (H) HEK293T cells were transfected with Flag-EGFR and myc-his-UCH-L1 plasmids, and then subjected to immunoprecipitation with anti-Flag or anti-Myc antibodies. The lysates and immunoprecipitates were then blotted. (I) MCF-7/AdrR cells transfected with myc-his-UCH-L1 plasmid were subjected immunoprecipitation with anti-Myc antibodies. The lysates and immunoprecipitates were analyzed. (J) Endogenous UCH-L1 and EGFR proteins interact with one another in MCF-7/AdrR cells. Endogenous EGFR proteins were immunoprecipitated with the anti-EGFR antibody. Endogenous UCH-L1 was detected by WB. (K) HEK293T cells transfected with Flag-EGFR were lysed and lysates were incubated with GST or GST-UCH-L1-GSH-Sepharose. Proteins retained on Sepharose were blotted with the indicated antibodies. (L and M) HEK293T cells transfected with the indicated constructs were treated with MG132 (20μM) for 8 hours before harvest. EGFR was immunoprecipitated with anti-Flag antibodies and immunoblotted with anti-HA antibodies. (N) Ubiquitinated EGFR was purified from MG132-treated HEK293T cells and then incubated with purified GST or GST-UCH-L1 in vitro, and then blotted with anti-HA antibodies.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation, Incubation, Construct, Purification, In Vitro

24) Product Images from "EGFR has a tumor-promoting role in liver macrophages during hepatocellular carcinoma formation"

Article Title: EGFR has a tumor-promoting role in liver macrophages during hepatocellular carcinoma formation

Journal: Nature cell biology

doi: 10.1038/ncb3031

EGFR expression in Kupffer cells/liver macrophages promotes HCC development ( a, b ) Numbers of F4/80 + cells in tumors of mice (left, EGFR f/f : n=66 (4 mice), EGFR Δhep : n=54 (4 mice), EGFR f/f : n=37 (3 mice), EGFR ΔMx : n=36 HPF (4 mice)) and CCL2 serum levels in HCC mice (right, EGFR f/f : n=4, EGFR Δhep : n=7, EGFR f/f : n=4 and EGFR ΔMx : n=6 mice). ( c ) Representative PCR showing EGFR deletion in isolated hepatocytes (Hep) and Kupffer cells (KC) of control (f/Δ) and EGFR Δhep/Δmac mice. flox = not deleted (1.1kb) and Δ = deleted EGFR (0.5kb). ( d ) Representative livers (top, scale bar: 1cm) and H E stainings of sections of indicated genotypes (bottom, scale bars: 1mm) 63 weeks after tumor initiation. Dotted lines mark tumor nodules. Note: Tumors of EGFR f/f mice are bigger than in Fig. 2c, d , because the tumors were analyzed 27 weeks later due to a change in the genetic background of the mice. ( e ) Tumor mass in livers of EGFR f/f (n=10), EGFR Δhep (n=5), EGFR Δhep/Δmac (n=5) and EGFR Δmac (n=4) mice. Data ( a, b ) represent mean±s.e.m. Data ( e ) represent mean ±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p
Figure Legend Snippet: EGFR expression in Kupffer cells/liver macrophages promotes HCC development ( a, b ) Numbers of F4/80 + cells in tumors of mice (left, EGFR f/f : n=66 (4 mice), EGFR Δhep : n=54 (4 mice), EGFR f/f : n=37 (3 mice), EGFR ΔMx : n=36 HPF (4 mice)) and CCL2 serum levels in HCC mice (right, EGFR f/f : n=4, EGFR Δhep : n=7, EGFR f/f : n=4 and EGFR ΔMx : n=6 mice). ( c ) Representative PCR showing EGFR deletion in isolated hepatocytes (Hep) and Kupffer cells (KC) of control (f/Δ) and EGFR Δhep/Δmac mice. flox = not deleted (1.1kb) and Δ = deleted EGFR (0.5kb). ( d ) Representative livers (top, scale bar: 1cm) and H E stainings of sections of indicated genotypes (bottom, scale bars: 1mm) 63 weeks after tumor initiation. Dotted lines mark tumor nodules. Note: Tumors of EGFR f/f mice are bigger than in Fig. 2c, d , because the tumors were analyzed 27 weeks later due to a change in the genetic background of the mice. ( e ) Tumor mass in livers of EGFR f/f (n=10), EGFR Δhep (n=5), EGFR Δhep/Δmac (n=5) and EGFR Δmac (n=4) mice. Data ( a, b ) represent mean±s.e.m. Data ( e ) represent mean ±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

Techniques Used: Expressing, Mouse Assay, Polymerase Chain Reaction, Isolation

EGFR expression is induced in activated Kupffer cells/liver macrophages under pathological conditions ( a-b ) Representative immunofluorescent confocal image showing co-staining for F4/80 and EGFR in cultured Kupffer cells/liver macrophages isolated from ( a ) EGFR f/f and ( b ) EGFR Δmac livers and stimulated with IL-1β for 24 h. Cultures contained ≥ 98% Kupffer cells/liver macrophages as confirmed by F4/80 staining. Scale bar: 50μm. ( c ) Representative Western Blot showing EGFR expression in isolated hepatocytes and Kupffer cells of EGFR f/f , EGFR ΔMx , EGFR Δhep and EGFR Δmac mice. ( d-e ) Representative immunofluorescent confocal images showing F4/80 and EGFR expression in liver sections of ( d ) untreated and ( e ) DEN treated (5 days) EGFR f/f mice. White arrows indicate EGFR-positive Kupffer cells. Scale bar: 50μm. ( f-g ) Mean fluorescence intensity (mean FI) showing EGFR expression levels (Alexa 488, green) in ( f ) liver macrophages ( EGFR f/f untreated: EGFR negative (n=9), EGFR positive (n=10); EGFR f/f 5 days after DEN: EGFR negative (n=4), EGFR positive (n=26) and ( g ) hepatocytes ( EGFR f/f untreated (n=12), EGFR f/f 5 days after DEN (n=13). Analysis of stainings shown in ( d ) and ( e ). Two pooled independent experiments for ( f ) and ( g ). ( h-k ) Representative anti-EGFR ( h, i ) and anti-F4/80 ( j, k ) staining performed on serial sections of control ( h, j ) and EGFR Δhep/Δmac ( i, k ) HCC showing EGFR expression in tumor cells and co-expression of EGFR and F4/80 in Kupffer cells/liver macrophages of EGFR f/f HCC and no EGFR expression in EGFR Δhep/Δmac tumors. Scale bar: 50μm. ( a-b, d-e ) Nuclei (DAPI, blue), EGFR (Alexa 488, green) and F4/80 (Alexa 594, red), merge (bottom right). Data ( f-g ) represent mean±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p
Figure Legend Snippet: EGFR expression is induced in activated Kupffer cells/liver macrophages under pathological conditions ( a-b ) Representative immunofluorescent confocal image showing co-staining for F4/80 and EGFR in cultured Kupffer cells/liver macrophages isolated from ( a ) EGFR f/f and ( b ) EGFR Δmac livers and stimulated with IL-1β for 24 h. Cultures contained ≥ 98% Kupffer cells/liver macrophages as confirmed by F4/80 staining. Scale bar: 50μm. ( c ) Representative Western Blot showing EGFR expression in isolated hepatocytes and Kupffer cells of EGFR f/f , EGFR ΔMx , EGFR Δhep and EGFR Δmac mice. ( d-e ) Representative immunofluorescent confocal images showing F4/80 and EGFR expression in liver sections of ( d ) untreated and ( e ) DEN treated (5 days) EGFR f/f mice. White arrows indicate EGFR-positive Kupffer cells. Scale bar: 50μm. ( f-g ) Mean fluorescence intensity (mean FI) showing EGFR expression levels (Alexa 488, green) in ( f ) liver macrophages ( EGFR f/f untreated: EGFR negative (n=9), EGFR positive (n=10); EGFR f/f 5 days after DEN: EGFR negative (n=4), EGFR positive (n=26) and ( g ) hepatocytes ( EGFR f/f untreated (n=12), EGFR f/f 5 days after DEN (n=13). Analysis of stainings shown in ( d ) and ( e ). Two pooled independent experiments for ( f ) and ( g ). ( h-k ) Representative anti-EGFR ( h, i ) and anti-F4/80 ( j, k ) staining performed on serial sections of control ( h, j ) and EGFR Δhep/Δmac ( i, k ) HCC showing EGFR expression in tumor cells and co-expression of EGFR and F4/80 in Kupffer cells/liver macrophages of EGFR f/f HCC and no EGFR expression in EGFR Δhep/Δmac tumors. Scale bar: 50μm. ( a-b, d-e ) Nuclei (DAPI, blue), EGFR (Alexa 488, green) and F4/80 (Alexa 594, red), merge (bottom right). Data ( f-g ) represent mean±s.d. Student‘s t -test for independent samples and unequal variances was used to assess statistical significance (*p

Techniques Used: Expressing, Staining, Cell Culture, Isolation, Western Blot, Mouse Assay, Fluorescence

25) Product Images from "Overexpression of miR-145 in U87 cells reduces glioma cell malignant phenotype and promotes survival after in vivo implantation"

Article Title: Overexpression of miR-145 in U87 cells reduces glioma cell malignant phenotype and promotes survival after in vivo implantation

Journal: International Journal of Oncology

doi: 10.3892/ijo.2014.2807

Effect of miR-145 overexpression or downregulation on ADAM17/EGFR/ERK activation in U87 cells. miR-145 was reported to directly target ADAM17 and EGFR. ADAM17 and EGFR expression showed a significant decrease in U87 miR-145 cells and a significant increase in U87 zip-145 cells. Expression of p-Erk showed the same trend as ADAM17/EGFR, but expression of Erk was not significantly affected by miR-145 expression levels.
Figure Legend Snippet: Effect of miR-145 overexpression or downregulation on ADAM17/EGFR/ERK activation in U87 cells. miR-145 was reported to directly target ADAM17 and EGFR. ADAM17 and EGFR expression showed a significant decrease in U87 miR-145 cells and a significant increase in U87 zip-145 cells. Expression of p-Erk showed the same trend as ADAM17/EGFR, but expression of Erk was not significantly affected by miR-145 expression levels.

Techniques Used: Over Expression, Activation Assay, Expressing

26) Product Images from "Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines"

Article Title: Erk2 but not Erk1 regulates crosstalk between Met and EGFR in squamous cell carcinoma cell lines

Journal: Molecular Cancer

doi: 10.1186/s12943-015-0319-z

Paracrine interaction model between TAM, tumor cells and endothelial cells. TAMs and tumor-associated stromal fibroblasts release a variety of factors that support tumor growth and progression. HGF, one of these factors, prompts tumor cells to produce the EGFR ligand amphiregulin (AR). Importantly, once the tumor cells are activated by HGF, their EGF receptor cannot be activated by EGFR ligands anymore. However, tumor-associated endothelial cells express high levels of EGFR and have been shown to respond to EGFR activation [ 38 , 42 , 43 ]. Therefore, we propose, that the tumor vasculature represent a possible target for the produced EGFR ligands.
Figure Legend Snippet: Paracrine interaction model between TAM, tumor cells and endothelial cells. TAMs and tumor-associated stromal fibroblasts release a variety of factors that support tumor growth and progression. HGF, one of these factors, prompts tumor cells to produce the EGFR ligand amphiregulin (AR). Importantly, once the tumor cells are activated by HGF, their EGF receptor cannot be activated by EGFR ligands anymore. However, tumor-associated endothelial cells express high levels of EGFR and have been shown to respond to EGFR activation [ 38 , 42 , 43 ]. Therefore, we propose, that the tumor vasculature represent a possible target for the produced EGFR ligands.

Techniques Used: Activation Assay, Produced

Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease ( p
Figure Legend Snippet: Amphiregulin is an activator of EGFR and HER2. The CM of a monocytic cell line induces the release of amphiregulin in SCC9 cells. (A) EGFR IP followed by Western blot analysis of SCC9 cells treated with 100 ng/ml HGF for 24 h and with 10 ng/ml EGF for 3 min. Immunoblots for phospho-tyrosine (=pY) and EGFR are shown. Total cell lysate blotted with tubulin was used as loading control. (B) EGFR and HER2 IP followed by Western blot analysis. SCC9 cells were stimulated for 5 min with HGF CM in the presence of 5 μg/ml amphiregulin-blocking antibody (=B-AR). The blocking antibody was added to the CM 30 min prior to stimulation. Immunoblots for pY, EGFR and HER2 are shown. (C) The CM of the monocytic cell line MAD-NT induced HGF-dependent amphiregulin release in SCC9 cells. Amphiregulin release was blocked, both when MAD-NT CM was pretreated with a HGF-blocking antibody (=B-HGF), as well as when SCC9 cells were pretreated for 30 min with 1 μm of the Met inhibitor PHA-665752 (=PHA). Ligand release was assayed using sandwich ELISA. Error bars indicate SEM of three independent experiments. The asterisk indicate a statistically significant decrease ( p

Techniques Used: Western Blot, Blocking Assay, Sandwich ELISA

27) Product Images from "Implication of calcium activated RasGRF2 in Annexin A6-mediated breast tumor cell growth and motility"

Article Title: Implication of calcium activated RasGRF2 in Annexin A6-mediated breast tumor cell growth and motility

Journal: Oncotarget

doi: 10.18632/oncotarget.26512

Down-regulation of RasGRF2 and inhibition of EGFR potently blocks the growth of TNBC cells ( A ) RNAi mediated down-regulation of RasGRF2 in AnxA6 expressing BT-549 cells. Cells were transfected with pools of 4 control non silencing siRNA (NSC-si) or 4 siRNAs targeting the coding sequence of RasGRF2 (GRF2-si). Cells were harvested and the down-regulation of RasGRF2 assessed by western blotting (left panel) and densitometric analysis of the protein band (right panel). ( B ) Clonogenic assays of control and RasGRF2 down-regulated cells. After 5 days in culture, control NSC-si and GRF2-si transfected cells were stained using crystal violet (0.5% in 1% acetic acid) and the growth of the cells was assessed by the number of colonies. ( C ) Control NSC-si and GRF2-si transfected cells were grown in growth factor reduced Matrigel 3D cultures for up to 7 days in the presence or absence of lapatinib (2 μM). Cell growth/viability was assessed using the PrestoBlue cell viability reagent and images of colonies were captured digitally. * indicates p
Figure Legend Snippet: Down-regulation of RasGRF2 and inhibition of EGFR potently blocks the growth of TNBC cells ( A ) RNAi mediated down-regulation of RasGRF2 in AnxA6 expressing BT-549 cells. Cells were transfected with pools of 4 control non silencing siRNA (NSC-si) or 4 siRNAs targeting the coding sequence of RasGRF2 (GRF2-si). Cells were harvested and the down-regulation of RasGRF2 assessed by western blotting (left panel) and densitometric analysis of the protein band (right panel). ( B ) Clonogenic assays of control and RasGRF2 down-regulated cells. After 5 days in culture, control NSC-si and GRF2-si transfected cells were stained using crystal violet (0.5% in 1% acetic acid) and the growth of the cells was assessed by the number of colonies. ( C ) Control NSC-si and GRF2-si transfected cells were grown in growth factor reduced Matrigel 3D cultures for up to 7 days in the presence or absence of lapatinib (2 μM). Cell growth/viability was assessed using the PrestoBlue cell viability reagent and images of colonies were captured digitally. * indicates p

Techniques Used: Inhibition, Expressing, Transfection, Sequencing, Western Blot, Staining

Loss of AnxA6 in invasive breast cancer cells is associated with early onset and rapid xenograft tumor growth ( A – C ) Control AnxA6 expressing BT-NSC, AnxA6-depleted BT-A6sh5 and AnxA6 deficient BT-A6A cells (1 × 10 6 /mouse) were implanted into mammary fat pads of 6-7 weeks old Nu/J mice ( n = 8). The growth of the xenograft tumors was monitored over time (A) and tumor size and weight (B and C) were determined following euthanasia of the tumor bearing mice. ( D ) Nu/J mice were injected with the indicated numbers of AnxA6-deficient BT-A6A cells and tumor volume was monitored as in (A) above. ( E – F ) Immunohistochemistry of xenograft tumors. (E) Formalin fixed, paraffin embedded thin sections of xenograft tumor tissues derived from AnxA6 down-regulated BT-A6sh5 and AnxA6 deficient BT-A6A cells were stained with antibodies against AnxA6, EGFR and RasGRF2 as well as with hematoxylin-eosin. (F) Immunostained tumor tissue sections were digitally scanned and quantified using the Tissue IA software (Leica Microsystems). ** indicates p
Figure Legend Snippet: Loss of AnxA6 in invasive breast cancer cells is associated with early onset and rapid xenograft tumor growth ( A – C ) Control AnxA6 expressing BT-NSC, AnxA6-depleted BT-A6sh5 and AnxA6 deficient BT-A6A cells (1 × 10 6 /mouse) were implanted into mammary fat pads of 6-7 weeks old Nu/J mice ( n = 8). The growth of the xenograft tumors was monitored over time (A) and tumor size and weight (B and C) were determined following euthanasia of the tumor bearing mice. ( D ) Nu/J mice were injected with the indicated numbers of AnxA6-deficient BT-A6A cells and tumor volume was monitored as in (A) above. ( E – F ) Immunohistochemistry of xenograft tumors. (E) Formalin fixed, paraffin embedded thin sections of xenograft tumor tissues derived from AnxA6 down-regulated BT-A6sh5 and AnxA6 deficient BT-A6A cells were stained with antibodies against AnxA6, EGFR and RasGRF2 as well as with hematoxylin-eosin. (F) Immunostained tumor tissue sections were digitally scanned and quantified using the Tissue IA software (Leica Microsystems). ** indicates p

Techniques Used: Expressing, Mouse Assay, Injection, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Derivative Assay, Staining, IA, Software

Down-regulation of RasGRF2 requires activation of EGFR and Ca 2+ influx from extracellular milieu ( A ) MDA-MB-468 cells were treated with or without EGF (50 ng/ml) following a time course and the cellular levels of activated EGFR (phospho-Y1068), RasGRF2, phospho-ERK1/2 and β-actin (loading control) assessed by western blotting; ( B ) Densitometric analysis of the cellular levels of these proteins over the 90 min time course for a representative experiment. ( C ) Cells were pretreated with BAPTA or EGTA for 1 h, then stimulated with EGF (50 ng/ml) in the presence of the respective Ca 2+ chelators for the indicated times. The expression of phospho-EGFR, RasGRF2 and Phospho-ERK1/2 was assessed by western blotting. ( D ) Densitometric analysis of the cellular levels of these proteins over the 30 min time course for a representative experiment. Experiments were repeated at least three times with similar results.
Figure Legend Snippet: Down-regulation of RasGRF2 requires activation of EGFR and Ca 2+ influx from extracellular milieu ( A ) MDA-MB-468 cells were treated with or without EGF (50 ng/ml) following a time course and the cellular levels of activated EGFR (phospho-Y1068), RasGRF2, phospho-ERK1/2 and β-actin (loading control) assessed by western blotting; ( B ) Densitometric analysis of the cellular levels of these proteins over the 90 min time course for a representative experiment. ( C ) Cells were pretreated with BAPTA or EGTA for 1 h, then stimulated with EGF (50 ng/ml) in the presence of the respective Ca 2+ chelators for the indicated times. The expression of phospho-EGFR, RasGRF2 and Phospho-ERK1/2 was assessed by western blotting. ( D ) Densitometric analysis of the cellular levels of these proteins over the 30 min time course for a representative experiment. Experiments were repeated at least three times with similar results.

Techniques Used: Activation Assay, Multiple Displacement Amplification, Western Blot, Expressing

28) Product Images from "EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling"

Article Title: EGFR is required for FOS‐dependent bone tumor development via RSK2/CREB signaling

Journal: EMBO Molecular Medicine

doi: 10.15252/emmm.201809408

EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection A Western blot analysis showing EGFR and c‐Fos protein levels in human OS cell lines cultured under standard conditions. B Cell viability of 143b or LM7 cells cultured for 24 h in medium (+10% FCS) with DMSO (1:2,000) or erlotinib (10 μM; n = 3, representative result from three independent experiments, shown as fold change, normalized to DMSO). C Cell viability of 143b or LM7 cells cultured for 24 h in medium (+0.5% FCS) containing 1× PBS (contr., 1:200) or EGF (50 ng/ml; n = 3, representative result from three independent experiments, shown as fold change, normalized to control). D Treatment scheme: 143b OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 14 days with vehicle or erlotinib (50 mg/kg) 7 days after injection, when the tumors started to grow. E 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle, 6 erlotinib; two independent experiments). F 143b tumor weight at endpoint ( n = 8 vehicle, 6 erlotinib; two independent experiments). G Treatment scheme: LM7 OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 21 days with vehicle or erlotinib (50 mg/kg) 42 days after injection, when the tumors started to grow. H LM7 xenograft growth curve during therapeutic regime ( n = 7 vehicle, 5 erlotinib; two independent experiments). I LM7 tumor weight at endpoint ( n = 7 vehicle, 5 erlotinib, two independent experiments). J IHC analysis of PCNA and cleaved caspase‐3 in 143b‐derived tumors ( n = 4). Scale bars: 50 μm. K IHC analysis of pCREB and c‐Fos in 143b‐derived tumors ( n = 5 for pCREB and n = 4 for c‐Fos analysis). Scale bars: 50 μm. L Western blot analysis of pCREB/CREB and p‐c‐Fos/c‐Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or erlotinib treatment. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B, C, F, I, J, and K) or by two‐way ANOVA followed by Bonferroni multiple comparison test (E and H). Source data are available online for this figure.
Figure Legend Snippet: EGFR inhibition decreases tumor progression of human OS cells 143b after orthotopic injection A Western blot analysis showing EGFR and c‐Fos protein levels in human OS cell lines cultured under standard conditions. B Cell viability of 143b or LM7 cells cultured for 24 h in medium (+10% FCS) with DMSO (1:2,000) or erlotinib (10 μM; n = 3, representative result from three independent experiments, shown as fold change, normalized to DMSO). C Cell viability of 143b or LM7 cells cultured for 24 h in medium (+0.5% FCS) containing 1× PBS (contr., 1:200) or EGF (50 ng/ml; n = 3, representative result from three independent experiments, shown as fold change, normalized to control). D Treatment scheme: 143b OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 14 days with vehicle or erlotinib (50 mg/kg) 7 days after injection, when the tumors started to grow. E 143b xenograft growth curve during therapeutic regime ( n = 8 vehicle, 6 erlotinib; two independent experiments). F 143b tumor weight at endpoint ( n = 8 vehicle, 6 erlotinib; two independent experiments). G Treatment scheme: LM7 OS cells (10 6 in 25 μl PBS) were intratibially injected; mice were treated for 21 days with vehicle or erlotinib (50 mg/kg) 42 days after injection, when the tumors started to grow. H LM7 xenograft growth curve during therapeutic regime ( n = 7 vehicle, 5 erlotinib; two independent experiments). I LM7 tumor weight at endpoint ( n = 7 vehicle, 5 erlotinib, two independent experiments). J IHC analysis of PCNA and cleaved caspase‐3 in 143b‐derived tumors ( n = 4). Scale bars: 50 μm. K IHC analysis of pCREB and c‐Fos in 143b‐derived tumors ( n = 5 for pCREB and n = 4 for c‐Fos analysis). Scale bars: 50 μm. L Western blot analysis of pCREB/CREB and p‐c‐Fos/c‐Fos protein expression in lysates directly isolated from 143b xenografts at endpoint, after 14 days of vehicle or erlotinib treatment. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B, C, F, I, J, and K) or by two‐way ANOVA followed by Bonferroni multiple comparison test (E and H). Source data are available online for this figure.

Techniques Used: Inhibition, Injection, Western Blot, Cell Culture, Mouse Assay, Immunohistochemistry, Derivative Assay, Expressing, Isolation, Two Tailed Test

EGFR signaling is essential for c‐Fos‐dependent bone tumor formation A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR/Egfr wa/+ and H2 ‐c‐fos LTR/Egfr wa/wa littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 16 wa/+, 11 wa/wa). E X‐ray analysis before (age = 2 months) and after (age = 7 months) vehicle or erlotinib treatment. Scale bars: 1 cm. F Quantification of tumor number during treatment ( n = 6 vehicle, 5 erlotinib). G Tumor size during treatment ( n = 6 vehicle, 5 erlotinib). H Analysis of serum ALP levels during treatment ( n = 5). I PET summation images (0–90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [ 11 C]erlotinib distribution in one H2‐ c‐fos LTR/ Egfr wt mouse (M125). Anatomical structures are labeled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1 cm. J Concentration–time curves of [ 11 C]erlotinib in bone tumors in right scapula of three H2‐ c‐fos LTR/ Egfr wt mice measured with PET. Broken line indicates threshold for in vitro effect of erlotinib (1 μM). Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B–D) or by two‐way ANOVA followed by Bonferroni multiple comparison test (F–H).
Figure Legend Snippet: EGFR signaling is essential for c‐Fos‐dependent bone tumor formation A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR/Egfr wa/+ and H2 ‐c‐fos LTR/Egfr wa/wa littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 23 wa/+, 15 wa/wa). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 16 wa/+, 11 wa/wa). E X‐ray analysis before (age = 2 months) and after (age = 7 months) vehicle or erlotinib treatment. Scale bars: 1 cm. F Quantification of tumor number during treatment ( n = 6 vehicle, 5 erlotinib). G Tumor size during treatment ( n = 6 vehicle, 5 erlotinib). H Analysis of serum ALP levels during treatment ( n = 5). I PET summation images (0–90 min) in horizontal (upper panel) and coronal view (lower panel) depicting [ 11 C]erlotinib distribution in one H2‐ c‐fos LTR/ Egfr wt mouse (M125). Anatomical structures are labeled with arrows (T, tumor; L, liver; H, heart; B, brain). Scale bars: 1 cm. J Concentration–time curves of [ 11 C]erlotinib in bone tumors in right scapula of three H2‐ c‐fos LTR/ Egfr wt mice measured with PET. Broken line indicates threshold for in vitro effect of erlotinib (1 μM). Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test (B–D) or by two‐way ANOVA followed by Bonferroni multiple comparison test (F–H).

Techniques Used: ALP Assay, Positron Emission Tomography, Labeling, Concentration Assay, Mouse Assay, In Vitro, Two Tailed Test

EGFR controls c‐Fos via MAPK‐dependent CREB activation in primary OS cells A Western blot analysis of primary OS cells isolated from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. B c‐fos and transgenic c‐fos ( c fos tg ) mRNA expression levels in primary H2‐ c‐fos LTR OS cells after 4× in vitro passages, cultured under standard conditions ( n = 3 independent cell lines). C Western blot analysis of H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib. D c‐fos and c‐fos tg mRNA expression levels in H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib (10 μM) or DMSO as control ( n = 4 independent cell lines). E, F Western blot analysis of starved H2 ‐c‐fos LTR/Egfr wt OS cells, pre‐treated with DMSO (1:1,000), afatinib (5 μM), GSK2233470 (10 μM), rapamycin (10 nM), or U0126 (10 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. G Western blot analysis of primary OS cells isolated from a p53 f/f Rb1 f/f Osx ‐Cre mouse after 24‐h erlotinib treatment. H c‐fos mRNA expression levels in p53 f/f Rb1 f/f Osx ‐Cre OS cells treated for 24 h with erlotinib (10 μM; n = 3). I Western blot analysis of starved p53 f/f Rb1 f/f Osx ‐Cre OS cells, pre‐treated with DMSO (1:1,000) or afatinib (5 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.
Figure Legend Snippet: EGFR controls c‐Fos via MAPK‐dependent CREB activation in primary OS cells A Western blot analysis of primary OS cells isolated from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. B c‐fos and transgenic c‐fos ( c fos tg ) mRNA expression levels in primary H2‐ c‐fos LTR OS cells after 4× in vitro passages, cultured under standard conditions ( n = 3 independent cell lines). C Western blot analysis of H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib. D c‐fos and c‐fos tg mRNA expression levels in H2 ‐c‐fos LTR/Egfr wt OS cells treated for 24 h with erlotinib (10 μM) or DMSO as control ( n = 4 independent cell lines). E, F Western blot analysis of starved H2 ‐c‐fos LTR/Egfr wt OS cells, pre‐treated with DMSO (1:1,000), afatinib (5 μM), GSK2233470 (10 μM), rapamycin (10 nM), or U0126 (10 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. G Western blot analysis of primary OS cells isolated from a p53 f/f Rb1 f/f Osx ‐Cre mouse after 24‐h erlotinib treatment. H c‐fos mRNA expression levels in p53 f/f Rb1 f/f Osx ‐Cre OS cells treated for 24 h with erlotinib (10 μM; n = 3). I Western blot analysis of starved p53 f/f Rb1 f/f Osx ‐Cre OS cells, pre‐treated with DMSO (1:1,000) or afatinib (5 μM) for 30 min and stimulated with EGF (50 ng/ml) as indicated. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

Techniques Used: Activation Assay, Western Blot, Isolation, Mouse Assay, Transgenic Assay, Expressing, In Vitro, Cell Culture, Two Tailed Test

Osteoblast‐specific EGFR deletion reduces c‐Fos‐driven OS formation A X‐ray analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 6–7 months of age ( n = 22 wt, 11 ΔOb). C Quantification of tumor size in tibiae ( n = 22 wt, 11 ΔOb). D ALP levels in the serum at 7‐month endpoint ( n = 17 wt, 8 ΔOb). E μPET/CT analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. F Standardized uptake values (SUV) of the μPET tracer Na[ 18 F]F in the pelvic OS of 4‐ and 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. n = 6 wt, 4 ΔOb for 4‐month time‐point, n = 6 wt, 3 ΔOb for 7‐month time‐point. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.
Figure Legend Snippet: Osteoblast‐specific EGFR deletion reduces c‐Fos‐driven OS formation A X‐ray analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 6–7 months of age ( n = 22 wt, 11 ΔOb). C Quantification of tumor size in tibiae ( n = 22 wt, 11 ΔOb). D ALP levels in the serum at 7‐month endpoint ( n = 17 wt, 8 ΔOb). E μPET/CT analysis of 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. F Standardized uptake values (SUV) of the μPET tracer Na[ 18 F]F in the pelvic OS of 4‐ and 7‐month‐old H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice. n = 6 wt, 4 ΔOb for 4‐month time‐point, n = 6 wt, 3 ΔOb for 7‐month time‐point. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

Techniques Used: ALP Assay, Mouse Assay, Two Tailed Test

EGFR is essential for proliferation, survival and c‐Fos protein and mRNA expression via RSK2/CREB phosphorylation A PCNA and cleaved caspase‐3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm 2 (for cleaved caspase‐3) in OS from H2 ‐c‐fos LTR/Egfr wt ( n = 6) and H2 ‐c‐fos LTR/Egfr ΔOb ( n = 5) mice. Scale bars: 100 μm. B Egfr , Ccnd1 , c‐fos and transgenic c‐fos ( c‐fos tg ) mRNA expression levels in tumors of H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice normalized to Tbp . n = 17 wt, 14 ΔOb ( Egfr, Ccnd1 ), n = 16 wt, 13 ΔOb ( c‐fos ), n = 16 wt, 14 ΔOb ( c‐fos tg ). C IHC staining and quantification showing pRSK2‐ ( n = 4), pCREB‐ ( n = 7 wt, 6 ΔOb), and c‐Fos ( n = 5)‐positive cells (%) in OS from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 100 μm. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.
Figure Legend Snippet: EGFR is essential for proliferation, survival and c‐Fos protein and mRNA expression via RSK2/CREB phosphorylation A PCNA and cleaved caspase‐3 IHC staining and quantification shown as % positive cells (for PCNA) and as positive cells per mm 2 (for cleaved caspase‐3) in OS from H2 ‐c‐fos LTR/Egfr wt ( n = 6) and H2 ‐c‐fos LTR/Egfr ΔOb ( n = 5) mice. Scale bars: 100 μm. B Egfr , Ccnd1 , c‐fos and transgenic c‐fos ( c‐fos tg ) mRNA expression levels in tumors of H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb mice normalized to Tbp . n = 17 wt, 14 ΔOb ( Egfr, Ccnd1 ), n = 16 wt, 13 ΔOb ( c‐fos ), n = 16 wt, 14 ΔOb ( c‐fos tg ). C IHC staining and quantification showing pRSK2‐ ( n = 4), pCREB‐ ( n = 7 wt, 6 ΔOb), and c‐Fos ( n = 5)‐positive cells (%) in OS from H2 ‐c‐fos LTR/Egfr wt and H2 ‐c‐fos LTR/Egfr ΔOb littermates. Scale bars: 100 μm. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test.

Techniques Used: Expressing, Immunohistochemistry, Staining, Mouse Assay, Transgenic Assay, Two Tailed Test

Osteoblast‐specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2 ‐c‐fos LTR mice A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR and H2‐ c‐fos LTR/ ColAREG littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 22 wt, 13 ColAREG ). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 22 wt, 13 ColAREG ). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 29 wt, 19 ColAREG ). E c‐fos mRNA expression levels in OSs of H2 ‐c‐fos LTR ( n = 11) and H2 ‐c‐fos LTR/ ColAREG mice ( n = 14). F, G Western blot analysis of bone tumor protein lysates from H2 ‐c‐fos LTR and H2 ‐c‐fos LTR/ ColAREG mice. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.
Figure Legend Snippet: Osteoblast‐specific overexpression of the EGFR ligand Amphiregulin accelerates tumor formation in H2 ‐c‐fos LTR mice A X‐ray analysis of 6‐month‐old H2 ‐c‐fos LTR and H2‐ c‐fos LTR/ ColAREG littermates. Scale bars: 1 cm. B Bone tumor number per mouse at 5–6 months of age ( n = 22 wt, 13 ColAREG ). C Quantification of tumor size in tibiae at 5–6 months of age ( n = 22 wt, 13 ColAREG ). D Alkaline phosphatase (ALP) levels in the serum at endpoint (age = 5–6 months; n = 29 wt, 19 ColAREG ). E c‐fos mRNA expression levels in OSs of H2 ‐c‐fos LTR ( n = 11) and H2 ‐c‐fos LTR/ ColAREG mice ( n = 14). F, G Western blot analysis of bone tumor protein lysates from H2 ‐c‐fos LTR and H2 ‐c‐fos LTR/ ColAREG mice. Data information: Data are shown as mean ± SEM. P ‐values were calculated by unpaired, two‐tailed t ‐test. Source data are available online for this figure.

Techniques Used: Over Expression, Mouse Assay, ALP Assay, Expressing, Western Blot, Two Tailed Test

EGFR and c‐Fos co‐expression in human OS negatively correlates with patient survival A Representative images of OS biopsies stained with antibodies against EGFR and c‐Fos. Scale bars: 50 μm. B Kaplan–Meier survival curve comparing the survival of patients with EGFR and c‐Fos double‐positive OSs against patients without co‐expression of both proteins ( n = 52). C Gene expression correlation analysis of several cancer‐associated RTKs with FOS in human OS (data analyzed from publicly available dataset E‐GEOD‐39058). Data information: P ‐values were calculated by log‐rank (Mantel–Cox) test comparing the two Kaplan–Meier curves (B) or by unpaired, two‐tailed t ‐test (C).
Figure Legend Snippet: EGFR and c‐Fos co‐expression in human OS negatively correlates with patient survival A Representative images of OS biopsies stained with antibodies against EGFR and c‐Fos. Scale bars: 50 μm. B Kaplan–Meier survival curve comparing the survival of patients with EGFR and c‐Fos double‐positive OSs against patients without co‐expression of both proteins ( n = 52). C Gene expression correlation analysis of several cancer‐associated RTKs with FOS in human OS (data analyzed from publicly available dataset E‐GEOD‐39058). Data information: P ‐values were calculated by log‐rank (Mantel–Cox) test comparing the two Kaplan–Meier curves (B) or by unpaired, two‐tailed t ‐test (C).

Techniques Used: Expressing, Staining, Two Tailed Test

29) Product Images from "mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes"

Article Title: mMAPS: A Flow-Proteometric Technique to Analyze Protein-Protein Interactions in Individual Signaling Complexes

Journal: Science signaling

doi: 10.1126/scisignal.2004620

mMAPS analysis of endogenous EGFR, STAT3, and associated complexes in tumor tissue sample
Figure Legend Snippet: mMAPS analysis of endogenous EGFR, STAT3, and associated complexes in tumor tissue sample

Techniques Used:

30) Product Images from "Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma"

Article Title: Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma

Journal: Genes & Development

doi: 10.1101/gad.1158703

Expression of Egfr and Her2 in pancreatic adenocarcinomas. ( A,B ) Immunohistochemistry with anti-Egfr ( A ) or anti-Her2 ( B ) antibodies shows robust expression of these proteins in the glandular regions of the tumors. ( C,D ) Immunohistochemistry for Egfr and Her2 reveals very weak or absent expression in the poorly differentiated regions of these tumors. Note that C and D were photographed from adjacent regions of the slides depicted in A and B .
Figure Legend Snippet: Expression of Egfr and Her2 in pancreatic adenocarcinomas. ( A,B ) Immunohistochemistry with anti-Egfr ( A ) or anti-Her2 ( B ) antibodies shows robust expression of these proteins in the glandular regions of the tumors. ( C,D ) Immunohistochemistry for Egfr and Her2 reveals very weak or absent expression in the poorly differentiated regions of these tumors. Note that C and D were photographed from adjacent regions of the slides depicted in A and B .

Techniques Used: Expressing, Immunohistochemistry

31) Product Images from "Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification"

Article Title: Differential roles of trans-phosphorylated EGFR, HER2, HER3, and RET as heterodimerisation partners of MET in lung cancer with MET amplification

Journal: British Journal of Cancer

doi: 10.1038/bjc.2011.322

Effects of depletion of MET, EGFR, HER2, HER3, or RET on cell proliferation, apoptosis, and migration in lung cancer cells with MET amplification ( A ), EBC-1 and H1993 cells were transfected with MET, EGFR, HER2, HER3, RET, or nonspecific (Con) siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins. ( B ) Cells transfected as in ( A ) were evaluated for cell proliferation. Absorbance values were expressed as a percentage of that for cells transfected with a control siRNA. ( C ) Cells transfected as in ( A ) were evaluated for the proportion of apoptotic cells. Data are expressed as the percentage increase in the number of annexin V-positive cells relative to the corresponding value for cells transfected with a control siRNA. ( D ) Cells were transfected with the indicated siRNAs for 24 h and then transferred in serum-free medium to cell culture inserts of a transwell apparatus for 24 h. The number of cells that migrated toward complete medium was counted with the use of a light microscope. Data are expressed relative to the value for cells transfected with a control siRNA. Data in ( B – D ) are means±s.e. from three independent experiments.
Figure Legend Snippet: Effects of depletion of MET, EGFR, HER2, HER3, or RET on cell proliferation, apoptosis, and migration in lung cancer cells with MET amplification ( A ), EBC-1 and H1993 cells were transfected with MET, EGFR, HER2, HER3, RET, or nonspecific (Con) siRNAs for 72 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins. ( B ) Cells transfected as in ( A ) were evaluated for cell proliferation. Absorbance values were expressed as a percentage of that for cells transfected with a control siRNA. ( C ) Cells transfected as in ( A ) were evaluated for the proportion of apoptotic cells. Data are expressed as the percentage increase in the number of annexin V-positive cells relative to the corresponding value for cells transfected with a control siRNA. ( D ) Cells were transfected with the indicated siRNAs for 24 h and then transferred in serum-free medium to cell culture inserts of a transwell apparatus for 24 h. The number of cells that migrated toward complete medium was counted with the use of a light microscope. Data are expressed relative to the value for cells transfected with a control siRNA. Data in ( B – D ) are means±s.e. from three independent experiments.

Techniques Used: Migration, Amplification, Transfection, Cell Culture, Light Microscopy

Proposed model for the roles of signalling pathways activated by heterodimers of MET and either EGFR, HER2, HER3, or RET in lung cancer cells with MET amplification.
Figure Legend Snippet: Proposed model for the roles of signalling pathways activated by heterodimers of MET and either EGFR, HER2, HER3, or RET in lung cancer cells with MET amplification.

Techniques Used: Amplification

Phosphorylation of multiple RTKs in lung cancer cells with MET amplification. ( A ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence (control) or presence of PHA-665752 (500 n). Cell lysates were prepared and incubated with an RTK array for determination of the phosphorylation status of each enzyme. Each RTK is spotted in duplicate, and the pairs of dots in each corner of the array are positive controls. The numbered pairs of dots correspond to the indicated phosphorylated (p-) RTKs. ( B ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence or presence of PHA-665752 (PHA, 500 n), gefitinib (1 μ ), lapatinib (1 μ ), or vandetanib (1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total forms of MET, EGFR, HER2, HER3, RET, AKT, ERK, or STAT3 or with those to β -actin (loading control). ( C ) The indicated cell lines were deprived of serum for 24 h and then incubated for 2 h in the absence (Con) or presence of gefitinib (Gef, 1 μ ), lapatinib (Lap, 1 μ ), or vandetanib (Van, 1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins.
Figure Legend Snippet: Phosphorylation of multiple RTKs in lung cancer cells with MET amplification. ( A ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence (control) or presence of PHA-665752 (500 n). Cell lysates were prepared and incubated with an RTK array for determination of the phosphorylation status of each enzyme. Each RTK is spotted in duplicate, and the pairs of dots in each corner of the array are positive controls. The numbered pairs of dots correspond to the indicated phosphorylated (p-) RTKs. ( B ) EBC-1 and H1993 cells were deprived of serum for 24 h and then incubated for 2 h in the absence or presence of PHA-665752 (PHA, 500 n), gefitinib (1 μ ), lapatinib (1 μ ), or vandetanib (1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to phosphorylated or total forms of MET, EGFR, HER2, HER3, RET, AKT, ERK, or STAT3 or with those to β -actin (loading control). ( C ) The indicated cell lines were deprived of serum for 24 h and then incubated for 2 h in the absence (Con) or presence of gefitinib (Gef, 1 μ ), lapatinib (Lap, 1 μ ), or vandetanib (Van, 1 μ ), after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins.

Techniques Used: Amplification, Incubation

Association of MET with EGFR, HER2, HER3, and RET in lung cancer cells positive for MET amplification. ( A ) Serum-deprived EBC-1 and H1993 cells were incubated for 2 h in the absence or presence of PHA-665752 (500 n), lysed, and subjected to immunoprecipitation (IP) with antibodies to MET or control IgG. The resulting precipitates were subjected to immunoblot analysis with antibodies to the indicated proteins. ( B ) Serum-deprived cells were lysed and subjected to IP with antibodies to EGFR, to HER2, to HER3, or to RET, and the resulting precipitates were subjected to immunoblot analysis with antibodies to the indicated proteins.
Figure Legend Snippet: Association of MET with EGFR, HER2, HER3, and RET in lung cancer cells positive for MET amplification. ( A ) Serum-deprived EBC-1 and H1993 cells were incubated for 2 h in the absence or presence of PHA-665752 (500 n), lysed, and subjected to immunoprecipitation (IP) with antibodies to MET or control IgG. The resulting precipitates were subjected to immunoblot analysis with antibodies to the indicated proteins. ( B ) Serum-deprived cells were lysed and subjected to IP with antibodies to EGFR, to HER2, to HER3, or to RET, and the resulting precipitates were subjected to immunoblot analysis with antibodies to the indicated proteins.

Techniques Used: Amplification, Incubation, Immunoprecipitation

32) Product Images from "Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab"

Article Title: Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, cetuximab

Journal: Oncotarget

doi: 10.18632/oncotarget.8596

AdC68-CTB and Hu5-CTB inhibits cell proliferation by reduced activation of EGFR, ERK and MEK A. NCI-H508 and DiFi cells were grown to a density of 1 × 10 4 cells/well in 96-well microtiter plates and treated with indicated adenoviruses in doses varying from 10 7 to 10 9 vp. After 72 h, an MTT assay was performed to quantify cell viability. Values are expressed as mean ± SEM. Statistical analysis was performed by Student's t test (* p
Figure Legend Snippet: AdC68-CTB and Hu5-CTB inhibits cell proliferation by reduced activation of EGFR, ERK and MEK A. NCI-H508 and DiFi cells were grown to a density of 1 × 10 4 cells/well in 96-well microtiter plates and treated with indicated adenoviruses in doses varying from 10 7 to 10 9 vp. After 72 h, an MTT assay was performed to quantify cell viability. Values are expressed as mean ± SEM. Statistical analysis was performed by Student's t test (* p

Techniques Used: CtB Assay, Activation Assay, MTT Assay

33) Product Images from "NSCLC depend upon YAP expression and nuclear localization after acquiring resistance to EGFR inhibitors"

Article Title: NSCLC depend upon YAP expression and nuclear localization after acquiring resistance to EGFR inhibitors

Journal: Genes & Cancer

doi: 10.18632/genesandcancer.136

A Western Blot of HCC827 and H1975 parental and drug-resistant sub-lines characterisation EGFR fluctuated slightly from HCC827 parental and both ER and GR sub-lines. AXL expression was observed in the drug-resistant sub-lines along with EMT marker Vimentin and loss of E-Cadherin. YAP was observed in parental cells and was amplified in the drug-resistant cells. EGFR remained consistent between H1975 parental cells and H1975/OR sub-line. AXL expression was also observed in parental and appeared to increase in expression as cells acquired resistance to osimertinib. EMT marker vimentin was down-regulated but remained expressed as e-cadherin was lost in H1975/OR. Merlin expression was increased in H1975/OR sub-line with co-expression of YAP.
Figure Legend Snippet: A Western Blot of HCC827 and H1975 parental and drug-resistant sub-lines characterisation EGFR fluctuated slightly from HCC827 parental and both ER and GR sub-lines. AXL expression was observed in the drug-resistant sub-lines along with EMT marker Vimentin and loss of E-Cadherin. YAP was observed in parental cells and was amplified in the drug-resistant cells. EGFR remained consistent between H1975 parental cells and H1975/OR sub-line. AXL expression was also observed in parental and appeared to increase in expression as cells acquired resistance to osimertinib. EMT marker vimentin was down-regulated but remained expressed as e-cadherin was lost in H1975/OR. Merlin expression was increased in H1975/OR sub-line with co-expression of YAP.

Techniques Used: Western Blot, Expressing, Marker, Amplification

34) Product Images from "Integrative Genomics Implicates EGFR as a Downstream Mediator in NKX2-1 Amplified Non-Small Cell Lung Cancer"

Article Title: Integrative Genomics Implicates EGFR as a Downstream Mediator in NKX2-1 Amplified Non-Small Cell Lung Cancer

Journal: PLoS ONE

doi: 10.1371/journal.pone.0142061

Defining the NKX2-1 cistrome by ChIPseq. (A) Validating the NKX2-1 antibody for ChIP: ChIP-PCR identifies the known NKX2-1 binding site in the SFTPB promoter. Note the enrichment of SFTPB compared to an irrelevant gene (WNT5A). (B) Overlap among the four NSCLC cell lines of NKX2-1 binding site-associated genes (within 100Kb). (C) De novo motif analysis re-discovers the known NKX2-1 consensus binding motif, and identifies enrichment of other transcription factor binding motifs nearby NKX2-1 binding sites. (D) NKX2-1 binding peaks identified at the EGFR locus in H1819 cells. The two called peaks are identified by blue triangles, and supporting reads are shown in the close-up inset. Binding peaks at EGFR in other cell lines are shown in Figure D, Panel B in S2 File .
Figure Legend Snippet: Defining the NKX2-1 cistrome by ChIPseq. (A) Validating the NKX2-1 antibody for ChIP: ChIP-PCR identifies the known NKX2-1 binding site in the SFTPB promoter. Note the enrichment of SFTPB compared to an irrelevant gene (WNT5A). (B) Overlap among the four NSCLC cell lines of NKX2-1 binding site-associated genes (within 100Kb). (C) De novo motif analysis re-discovers the known NKX2-1 consensus binding motif, and identifies enrichment of other transcription factor binding motifs nearby NKX2-1 binding sites. (D) NKX2-1 binding peaks identified at the EGFR locus in H1819 cells. The two called peaks are identified by blue triangles, and supporting reads are shown in the close-up inset. Binding peaks at EGFR in other cell lines are shown in Figure D, Panel B in S2 File .

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction, Binding Assay

Combined NKX2-1 and EGFR knockdown reduces cell proliferation and MAPK/PI3K signaling. (A) Combined knockdown of NKX2-1 and EGFR reduces H1819 cell proliferation more than either alone. **, P -value
Figure Legend Snippet: Combined NKX2-1 and EGFR knockdown reduces cell proliferation and MAPK/PI3K signaling. (A) Combined knockdown of NKX2-1 and EGFR reduces H1819 cell proliferation more than either alone. **, P -value

Techniques Used:

NKX2-1 regulates EGFR levels, with negative feedback. (A) NKX2-1 knockdown leads to reduced EGFR protein levels quantified by western blot (% residual indicated). Levels normalized to α-tubulin loading control. (B) EGFR knockdown by siRNA reduces cell proliferation comparable to NKX2-1 knockdown (see Fig 1A ). **, P -value
Figure Legend Snippet: NKX2-1 regulates EGFR levels, with negative feedback. (A) NKX2-1 knockdown leads to reduced EGFR protein levels quantified by western blot (% residual indicated). Levels normalized to α-tubulin loading control. (B) EGFR knockdown by siRNA reduces cell proliferation comparable to NKX2-1 knockdown (see Fig 1A ). **, P -value

Techniques Used: Western Blot

Model of EGFR as downstream mediator NKX2-1 oncogenic signaling. Schematic figure summarizes pathways and relationships deduced from experimental findings; see discussion in main text.
Figure Legend Snippet: Model of EGFR as downstream mediator NKX2-1 oncogenic signaling. Schematic figure summarizes pathways and relationships deduced from experimental findings; see discussion in main text.

Techniques Used:

35) Product Images from "EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells"

Article Title: EGFR deficiency leads to impaired self-renewal and pluripotency of mouse embryonic stem cells

Journal: PeerJ

doi: 10.7717/peerj.6314

Inhibition of EGFR by AG1478 impairs mESC self-renewal and pluripotency, and induces differentiation. (A) Enzymatic activity for AP was analyzed in control and AG1478 treated mESCs. (B–D) IF staining against SSEA-1 (red), OCT-4 (green), and Nanog (red) in control and AG1478 treated mESCs. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole, scale bar: 200 μm. (E and F) Quantitative PCR analysis of mRNA levels of pluripotency factor s and differentiation related genes in control and AG1478 treated mESCs. The amounts of each mRNA were normalized to GAPDH mRNA and are shown relative to the amounts in control mESCs (set to 1). (G) Protein expression of OCT4 and GATA4 in control and AG1478 treated mESCs. β-actin served as a loading control. (H) Quantified relative band intensity ratio of OCT4 and GATA4. The data are presented as mean ± SD ( n = 3; ** P
Figure Legend Snippet: Inhibition of EGFR by AG1478 impairs mESC self-renewal and pluripotency, and induces differentiation. (A) Enzymatic activity for AP was analyzed in control and AG1478 treated mESCs. (B–D) IF staining against SSEA-1 (red), OCT-4 (green), and Nanog (red) in control and AG1478 treated mESCs. Nuclei were counterstained by 4′,6-diamidino-2-phenylindole, scale bar: 200 μm. (E and F) Quantitative PCR analysis of mRNA levels of pluripotency factor s and differentiation related genes in control and AG1478 treated mESCs. The amounts of each mRNA were normalized to GAPDH mRNA and are shown relative to the amounts in control mESCs (set to 1). (G) Protein expression of OCT4 and GATA4 in control and AG1478 treated mESCs. β-actin served as a loading control. (H) Quantified relative band intensity ratio of OCT4 and GATA4. The data are presented as mean ± SD ( n = 3; ** P

Techniques Used: Inhibition, Activity Assay, Staining, Real-time Polymerase Chain Reaction, Expressing

36) Product Images from "α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells"

Article Title: α2,3-sialyltransferase type I regulates migration and peritoneal dissemination of ovarian cancer cells

Journal: Oncotarget

doi: 10.18632/oncotarget.15994

ST3GalI interacts with EGFR signaling pathway in ovarian cancer (A) The correlation of 20 STs and significant cell receptors in the ES2ovarian cancer cell line were analyzed by the L1000 mRNA microarray. Data shown are mean ± SD of separate repeat experiments. (B) The analysis of mRNA expression of ST3GalI and EGFR from Oncomine TCGA (n=562), Bittner (n=241), and Lu (n=50) ovarian cancer genomics is shown. The expression of EGFR was compared between low and high ST3GalI groups using a tercile approach. (C) The mRNA expression of α2,3-sialyltransferases and TKI drug efficiency (Actarea) are shown from the CCLE ovarian cancer dataset. (D-E) Western blot analysis of EGFR and phospho-EGFR in ST3GalI knocked-down or overexpressing cells compared to controls is shown. GAPDH was used as control (same GAPDH as in Figure 1D-1E ). (F) The co-immunoprecipitation assay coupled with immunoblotting analysis to evaluate the protein-protein interaction of ST3GalI and EGFR is shown. (G) Immunoblotting of ST3Ga1I immunoprecipitated with anti-EGFR antibodies in SC and ST3GalI knock-down of ES2 cell line are shown. (H) Time course RNA and protein analysis showing EGFR expression during ST3GalI knock-down.
Figure Legend Snippet: ST3GalI interacts with EGFR signaling pathway in ovarian cancer (A) The correlation of 20 STs and significant cell receptors in the ES2ovarian cancer cell line were analyzed by the L1000 mRNA microarray. Data shown are mean ± SD of separate repeat experiments. (B) The analysis of mRNA expression of ST3GalI and EGFR from Oncomine TCGA (n=562), Bittner (n=241), and Lu (n=50) ovarian cancer genomics is shown. The expression of EGFR was compared between low and high ST3GalI groups using a tercile approach. (C) The mRNA expression of α2,3-sialyltransferases and TKI drug efficiency (Actarea) are shown from the CCLE ovarian cancer dataset. (D-E) Western blot analysis of EGFR and phospho-EGFR in ST3GalI knocked-down or overexpressing cells compared to controls is shown. GAPDH was used as control (same GAPDH as in Figure 1D-1E ). (F) The co-immunoprecipitation assay coupled with immunoblotting analysis to evaluate the protein-protein interaction of ST3GalI and EGFR is shown. (G) Immunoblotting of ST3Ga1I immunoprecipitated with anti-EGFR antibodies in SC and ST3GalI knock-down of ES2 cell line are shown. (H) Time course RNA and protein analysis showing EGFR expression during ST3GalI knock-down.

Techniques Used: Microarray, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

α2,3-sialylation inhibitor SsaI affects EGFR signaling and synergizes with TKI (A) Quantification of EGFR and phospho-EGFR in ES2 cells treated with 100μM SSaI or DMSO control for 72h; GAPDH was used as control (same GAPDH as in Figure 3 ). (B) Intraperitoneal tumors from B6 mice treated with either SsaI or DMSO control were stained by the Duolink in situ IHC staining kit to analyze for ST3GalI and EGFR. (C) ES2 cells were treated with 100μM SsaI and 5μm AG1478 (EGFR inhibitor) were subjected to Transwell matrigel invasion assay. Total numbers of cells were counted in 7 to 10 random fields. Data shown are the mean ± SD of 3 separate experiments. (D) Synergy between ST3GalI and EGFR were determined by treating ES2 cells with either SsaI orAG1478 (EGFR inhibitor) or both for 48h followed by determination of their effects on cell proliferation rate. Data shown are the mean ± SD of 3 independent experiments. (E) A proposed mechanism of the interaction between ST3GalI and the EGFR signaling pathway.
Figure Legend Snippet: α2,3-sialylation inhibitor SsaI affects EGFR signaling and synergizes with TKI (A) Quantification of EGFR and phospho-EGFR in ES2 cells treated with 100μM SSaI or DMSO control for 72h; GAPDH was used as control (same GAPDH as in Figure 3 ). (B) Intraperitoneal tumors from B6 mice treated with either SsaI or DMSO control were stained by the Duolink in situ IHC staining kit to analyze for ST3GalI and EGFR. (C) ES2 cells were treated with 100μM SsaI and 5μm AG1478 (EGFR inhibitor) were subjected to Transwell matrigel invasion assay. Total numbers of cells were counted in 7 to 10 random fields. Data shown are the mean ± SD of 3 separate experiments. (D) Synergy between ST3GalI and EGFR were determined by treating ES2 cells with either SsaI orAG1478 (EGFR inhibitor) or both for 48h followed by determination of their effects on cell proliferation rate. Data shown are the mean ± SD of 3 independent experiments. (E) A proposed mechanism of the interaction between ST3GalI and the EGFR signaling pathway.

Techniques Used: Mouse Assay, Staining, In Situ, Immunohistochemistry, Invasion Assay

37) Product Images from "TNFα promotes mucosal wound repair through enhanced Platelet Activating Factor Receptor signaling in the epithelium"

Article Title: TNFα promotes mucosal wound repair through enhanced Platelet Activating Factor Receptor signaling in the epithelium

Journal: Mucosal immunology

doi: 10.1038/s41385-019-0150-8

Schematic model illustrating the molecular mechanism of PAFR signaling during epithelial wound repair. Our working hypothesis is that in response to wounding TNFα/IFNγ signaling increases expression of PAFR . Binding of PAF leads to PAFR stimulation which causes Src phosphorylation and ADAM10 activation, inducing cleavage of EGFR ligands, such as HB-EGF, and EGFR activation. Stimulation of EGFR further enhances phosphorylation of Src, which subsequently activates Rac1 via GEFs, increasing ROS accumulation and phosphorylation of FAK. In parallel, Src activation induces FAK phosphorylation to further promote turnover of focal adhesion complexes leading to modulation of cellular migration.
Figure Legend Snippet: Schematic model illustrating the molecular mechanism of PAFR signaling during epithelial wound repair. Our working hypothesis is that in response to wounding TNFα/IFNγ signaling increases expression of PAFR . Binding of PAF leads to PAFR stimulation which causes Src phosphorylation and ADAM10 activation, inducing cleavage of EGFR ligands, such as HB-EGF, and EGFR activation. Stimulation of EGFR further enhances phosphorylation of Src, which subsequently activates Rac1 via GEFs, increasing ROS accumulation and phosphorylation of FAK. In parallel, Src activation induces FAK phosphorylation to further promote turnover of focal adhesion complexes leading to modulation of cellular migration.

Techniques Used: Expressing, Binding Assay, Activation Assay, Migration

38) Product Images from "Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma"

Article Title: Exosomal transfer of miR-1238 contributes to temozolomide-resistance in glioblastoma

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2019.03.016

A: Relative expression of miR-1238 levels after transfection of miR-NC or miR-1238 inhibitor in U251r and N3r cells. B: Relative expression of miR-1238 levels of NC exosomes and 1238-down exosomes. C: Parallel experiments were performed to evaluate the modulation of the EGFR-PI3K-Akt-mTOR pathway by miR-1238/CAV1 signaling in N3r cells. In all experiments, bars represent mean ± SD for three replicates. (Statistical analysis was performed by Student‘s t-test, *P
Figure Legend Snippet: A: Relative expression of miR-1238 levels after transfection of miR-NC or miR-1238 inhibitor in U251r and N3r cells. B: Relative expression of miR-1238 levels of NC exosomes and 1238-down exosomes. C: Parallel experiments were performed to evaluate the modulation of the EGFR-PI3K-Akt-mTOR pathway by miR-1238/CAV1 signaling in N3r cells. In all experiments, bars represent mean ± SD for three replicates. (Statistical analysis was performed by Student‘s t-test, *P

Techniques Used: Expressing, Transfection

MiR-1238/CAV1 signaling modulates TMZ resistance via EGFR pathway. A: Representative confocal images of CAV1 (green) and EGFR (red) colocalized in U251 and N3 cells (scale bars = 10 μm). B: Co-IP was performed using lysates from U251 and N3 cells transfected with FLAG-CAV1, Myc-EGFR, or vector control. Western blotting was performed with the indicated antibodies. C: U251 and N3 cells transfected with si-CAV1 or control were treated with EGF (50 ng/mL) for the times shown prior to harvest, lysis, and analysis by western blot using the antibodies indicated. D: Subcellular fractionation of U251r and N3r cells created 10 fractions and each was tested for the presence of CAV1 by western blotting. Fractions 3, 4, and 5 from U251r and 4, 5, and 6 from N3r were pooled and considered as caveolae membranes. E: Western blot analysis for phospho-EGFR, EGFR, CAV1, and PTEN in collected fractions or cell lysate of U251r cells. F: Western blot analysis was performed to estimate the activation of the PI3K-Akt-mTOR pathway in U251r cells transfected with miR-1238 inhibitor or shCAV1 with TMZ treatment or not. All results are representative of at least three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Figure Legend Snippet: MiR-1238/CAV1 signaling modulates TMZ resistance via EGFR pathway. A: Representative confocal images of CAV1 (green) and EGFR (red) colocalized in U251 and N3 cells (scale bars = 10 μm). B: Co-IP was performed using lysates from U251 and N3 cells transfected with FLAG-CAV1, Myc-EGFR, or vector control. Western blotting was performed with the indicated antibodies. C: U251 and N3 cells transfected with si-CAV1 or control were treated with EGF (50 ng/mL) for the times shown prior to harvest, lysis, and analysis by western blot using the antibodies indicated. D: Subcellular fractionation of U251r and N3r cells created 10 fractions and each was tested for the presence of CAV1 by western blotting. Fractions 3, 4, and 5 from U251r and 4, 5, and 6 from N3r were pooled and considered as caveolae membranes. E: Western blot analysis for phospho-EGFR, EGFR, CAV1, and PTEN in collected fractions or cell lysate of U251r cells. F: Western blot analysis was performed to estimate the activation of the PI3K-Akt-mTOR pathway in U251r cells transfected with miR-1238 inhibitor or shCAV1 with TMZ treatment or not. All results are representative of at least three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Techniques Used: Co-Immunoprecipitation Assay, Transfection, Plasmid Preparation, Western Blot, Lysis, Fractionation, Activation Assay

Relative expression of CAV1 and activation of the EGFR-PI3K-Akt-mTOR pathway in recipient cells after incubation with resistant exosomes.
Figure Legend Snippet: Relative expression of CAV1 and activation of the EGFR-PI3K-Akt-mTOR pathway in recipient cells after incubation with resistant exosomes.

Techniques Used: Expressing, Activation Assay, Incubation

39) Product Images from "Downregulation of BANCR Promotes Aggressiveness in Papillary Thyroid Cancer via the MAPK and PI3K Pathways"

Article Title: Downregulation of BANCR Promotes Aggressiveness in Papillary Thyroid Cancer via the MAPK and PI3K Pathways

Journal: Journal of Cancer

doi: 10.7150/jca.20150

The phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in BANCR -silenced or -overexpressing NPA and TPC1 cells was detected by western blotting. The loss of BANCR induced the phosphorylation of ERK1/2 (C). However, there was no activation of upstream molecule including EGFR (A), MEK (C), MEK, STAT-1, and STAT-3 (E). BANCR overexpression had no effect on the phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in either cell line (B, D and F). GAPDH expression was used to normalize protein loading.
Figure Legend Snippet: The phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in BANCR -silenced or -overexpressing NPA and TPC1 cells was detected by western blotting. The loss of BANCR induced the phosphorylation of ERK1/2 (C). However, there was no activation of upstream molecule including EGFR (A), MEK (C), MEK, STAT-1, and STAT-3 (E). BANCR overexpression had no effect on the phosphorylation of EGFR, MEK, ERK1/2, STAT-1, and STAT-3 in either cell line (B, D and F). GAPDH expression was used to normalize protein loading.

Techniques Used: Western Blot, Activation Assay, Over Expression, Expressing

40) Product Images from "PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules"

Article Title: PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2018.10.072

PPARγ suppresses glycolysis via inhibition of EGFR in PT cells. (A) NRK-52E cells with or without PPARγ depletion were transfected with a control vector or a vector expressing PPARγ. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (B, C) The media of serum-starved NRK-52E cells with or without PPARγ depletion and with or without reconstituted expression of PPARγ were collected for analysis of glucose consumption (B) and lactate production(C). The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (D) NRK-52E cells with or without PPARγ depletion were lysed. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR, P-ERK1/2, total-ERK1, P-AKT, Total-AKT. All the experiments data were performed in triplicate at least and the representative imagines were shown. (E) NRK-52E cells with or without PPARγ depletion were treated with or without EGFR inhibitor AG1478 (2 μM) for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, HK2, PFKL, GLUT1, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (F) NRK-52E cells with or without PPARγ overexpression were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT1, GAPDH, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (G, H) The media of serum-starved NRK-52E cells with or without PPARγ depletion in the presence or absence of AG1478 (2 μM) for 48 h were collected for analysis of glucose consumption (G) and lactate production (H). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05;** P ≤ .01; *** P ≤ .001.
Figure Legend Snippet: PPARγ suppresses glycolysis via inhibition of EGFR in PT cells. (A) NRK-52E cells with or without PPARγ depletion were transfected with a control vector or a vector expressing PPARγ. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (B, C) The media of serum-starved NRK-52E cells with or without PPARγ depletion and with or without reconstituted expression of PPARγ were collected for analysis of glucose consumption (B) and lactate production(C). The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (D) NRK-52E cells with or without PPARγ depletion were lysed. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR, P-ERK1/2, total-ERK1, P-AKT, Total-AKT. All the experiments data were performed in triplicate at least and the representative imagines were shown. (E) NRK-52E cells with or without PPARγ depletion were treated with or without EGFR inhibitor AG1478 (2 μM) for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, HK2, PFKL, GLUT1, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (F) NRK-52E cells with or without PPARγ overexpression were treated with or without EGF (100 ng/ml) for 24 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, HK2, PFKL, GLUT1, GAPDH, LDHA. All the experiments data were performed in triplicate at least and the representative imagines were shown. (G, H) The media of serum-starved NRK-52E cells with or without PPARγ depletion in the presence or absence of AG1478 (2 μM) for 48 h were collected for analysis of glucose consumption (G) and lactate production (H). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05;** P ≤ .01; *** P ≤ .001.

Techniques Used: Inhibition, Transfection, Plasmid Preparation, Expressing, Over Expression

PPARγ suppresses the glycolysis in PT through iRhom2-mediated downregulation of EGF expression and secretion. (A) Real time PCR analyses of mRNA levels of the indicated gene in NRK-52E cells with or without PPARγ depletion were performed. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .0 01. ns represents not significant difference between the indicated sample and the counterpart in the absence of PPARγ depletion. (B) The intracellular and secreted EGF levels of NRK-52E cells with or without PPARγ depletion were analyzed by immunoblotting assay with the indicated antibodies including PPARγ and EGF (left) and ELISA (right), respectively. * P ≤ .05. (C) NRK-52E cells with or without PPARγ depletion were treated with or without EGF neutralizing antibody for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR. All the experiments data were performed in triplicate at least and the representative imagines were shown. (D) The mRNA levels of PPAR γ and RHBDF2 of NRK-52E cells with or without PPARγ depletion were determined by real time PCR analysis. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (E, F) NRK-52E cells with or without PPARγ depletion were transfected with or without a vector expressing iRhom2. Immunoblotting analyses with the indicated antibodies including PPARγ, iRhom2, EGF, P-EGFR, Total-EGFR, P-ERK1/2, Total-ERK1, P-AKT, Total-AKT, HK2, PFKL, GLUT1, LDHA (E) and ELISA analyses of secreted EGF levels (F) were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01. (G) Sequence alignment of the putative PPRE within the human, rat, and mouse RHBDF2 promoters. The varied nucleotides in WT RHBDF2 promoter of different species and mutated nucleotides in the RHBDF2 promoter used for luciferase assay were labeled in red. (H) ChIP analyses of NRK-52E cells were performed with an anti-PPARγ antibody and the primers for the putative PPRE region of RHBDF2 promoter. (I) Luciferase reporter vectors with species-specific or mutated RHBDF2 promoter were co-transfected with the vector expressing PPARγ into HEK293FT cells treated with or without PPARγ ligand Rosiglitazone (TZD) (50 μM). Luciferase reporter analyses were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01; *** P ≤ .001. (J, K) Serum-starved NRK52-E cells with or without PPARγ depletion or iRhom2 overexpression were treated with or without EGF (100 ng/ml) for 24 h. The media were collected for analysis of glucose consumption (j) and lactate production (k). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05; ** P ≤ .01.
Figure Legend Snippet: PPARγ suppresses the glycolysis in PT through iRhom2-mediated downregulation of EGF expression and secretion. (A) Real time PCR analyses of mRNA levels of the indicated gene in NRK-52E cells with or without PPARγ depletion were performed. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .0 01. ns represents not significant difference between the indicated sample and the counterpart in the absence of PPARγ depletion. (B) The intracellular and secreted EGF levels of NRK-52E cells with or without PPARγ depletion were analyzed by immunoblotting assay with the indicated antibodies including PPARγ and EGF (left) and ELISA (right), respectively. * P ≤ .05. (C) NRK-52E cells with or without PPARγ depletion were treated with or without EGF neutralizing antibody for 48 h. Immunoblotting analyses were performed with the indicated antibodies including PPARγ, P-EGFR, Total-EGFR. All the experiments data were performed in triplicate at least and the representative imagines were shown. (D) The mRNA levels of PPAR γ and RHBDF2 of NRK-52E cells with or without PPARγ depletion were determined by real time PCR analysis. The data represent the mean ± SD from n = 3 independent experiments. *** P ≤ .001. (E, F) NRK-52E cells with or without PPARγ depletion were transfected with or without a vector expressing iRhom2. Immunoblotting analyses with the indicated antibodies including PPARγ, iRhom2, EGF, P-EGFR, Total-EGFR, P-ERK1/2, Total-ERK1, P-AKT, Total-AKT, HK2, PFKL, GLUT1, LDHA (E) and ELISA analyses of secreted EGF levels (F) were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01. (G) Sequence alignment of the putative PPRE within the human, rat, and mouse RHBDF2 promoters. The varied nucleotides in WT RHBDF2 promoter of different species and mutated nucleotides in the RHBDF2 promoter used for luciferase assay were labeled in red. (H) ChIP analyses of NRK-52E cells were performed with an anti-PPARγ antibody and the primers for the putative PPRE region of RHBDF2 promoter. (I) Luciferase reporter vectors with species-specific or mutated RHBDF2 promoter were co-transfected with the vector expressing PPARγ into HEK293FT cells treated with or without PPARγ ligand Rosiglitazone (TZD) (50 μM). Luciferase reporter analyses were performed. The data represent the mean ± SD from n = 3 independent experiments. ** P ≤ .01; *** P ≤ .001. (J, K) Serum-starved NRK52-E cells with or without PPARγ depletion or iRhom2 overexpression were treated with or without EGF (100 ng/ml) for 24 h. The media were collected for analysis of glucose consumption (j) and lactate production (k). The data represent the mean ± SD from n = 3 independent experiments. * P ≤ .05; ** P ≤ .01.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Sequencing, Luciferase, Labeling, Chromatin Immunoprecipitation, Over Expression

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    The inhibition of ERK1/2 phosphorylation and function by <t>EGFR</t> and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and <t>RSK1/2/3</t> as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P
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    Dephosphorylation of <t>EGFR</t> v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . <t>Akt</t> phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.
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    Validation of specific peptide binding to <t>EGFR.</t> On confocal microscopy, we found strong binding of ( a ) <t>QRH*-Cy5.5</t> peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.
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    The inhibition of ERK1/2 phosphorylation and function by EGFR and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and RSK1/2/3 as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P

    Journal: Carcinogenesis

    Article Title: Omega-3 polyunsaturated fatty acids selectively inhibit growth in neoplastic oral keratinocytes by differentially activating ERK1/2

    doi: 10.1093/carcin/bgt257

    Figure Lengend Snippet: The inhibition of ERK1/2 phosphorylation and function by EGFR and MEK antagonists. ( a ) Western blots showing the effect of the indicated antagonists on ERK1/2 phosphorylation and its downstream target P-p90RSK as induced by 3 µM DHA and EPA in addition to the ethanol control (CON) 2 h after treatment. Total ERK1/2 and RSK1/2/3 as well as GAPDH served as loading controls. The EGFR neutralizing antibody (EGFR-Ab) was used at 1 and 10 µg/ml; the EGFR kinase inhibitor AG1478 was used at 100 nM and 1 µM; and the MEK inhibitor AZD6244 was used at 100 and 300 nM. ( e ) Western blot comparing the effect of AG1478 with the pan-caspase inhibitor Q-VD-Oph on ERK1/2 as induced by 5 µM DHA and EPA in addition to the ethanol vehicle control 2 h after treatment. The blots are typical of three independent experiments. The graphs show the relative to the control mean value of P-ERK/total ERK ( b and f ) and P-p90RSK/total RSK ( c ), and they represent the mean values of three or more western blots measured by densitometry. ( d ) The graph shows the relative mean values of the n-3 PUFA- and AZD6244-treated cells compared with the untreated vehicle control. Significantly different from the mean value of the untreated vehicle control (* P

    Article Snippet: RSK1/2/3 and EGFR (both from Cell Signaling) and phospho-EGFR (BD Transduction Laboratories) antibodies were used at 1:1000 and 1:500, respectively.

    Techniques: Inhibition, Western Blot

    Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.

    Journal: Cancer Medicine

    Article Title: Nimotuzumab enhances temozolomide‐induced growth suppression of glioma cells expressing mutant EGFR in vivo

    doi: 10.1002/cam4.614

    Figure Lengend Snippet: Dephosphorylation of EGFR v III (∆ EGFR ) and wild‐type (wt) EGFR upon treatment with nimotuzumab or tyrphostin AG 1478 in vitro. Cultured human glioma U87 MG cells overexpressing either EGFR v III (U87 MG .∆ EGFR ) or wt EGFR (U87 MG .wt EGFR ) were treated with nimotuzumab for 72 h and their lysates were subjected to Western blot analysis. Nimotuzumab dephosphorylated EGFR at both tyrosine residues 1068 and 1173. EGFR v III tyrosine phosphorylation was preferentially suppressed by nimotuzumab at lower doses, compared with wild‐type EGFR . Akt phosphorylation at threonine residue 308 was modestly suppressed in U87 MG .∆ EGFR cells by nimotuzumab. Relative tyrosine phosphorylation per molecule is shown below each lane, calculated as a ratio of that of untreated status and standardized with actin expression. A tyrphostin AG 1478 was used as a positive control for EGFR inhibition.

    Article Snippet: Proteins on the PVDF membranes were probed with antibodies against EGFR (C13, BD Biosciences, San Jose, CA), P‐EGFR (Tyr1068), P‐EGFR (Tyr1173), Akt, P‐Akt (Thr308), P‐Akt (Ser473), P‐mammalian target of rapamycin (mTOR) (Ser2448), mTOR (Cell Signaling, Danvers, MA), O 6 ‐methylguanine‐DNA methyltransferase (MGMT) (MT3.1, Neo Markers, Fremont, CA), MSH6, MLH1, MSH2, PMS2 (BD Biosciences), detected by chemiluminescence, and quantified (ImageQuant LAS4010, GE Healthcare, Tokyo, Japan).

    Techniques: De-Phosphorylation Assay, In Vitro, Cell Culture, Western Blot, Expressing, Positive Control, Inhibition

    Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Validation of specific peptide binding to EGFR. On confocal microscopy, we found strong binding of ( a ) QRH*-Cy5.5 peptide (red) and ( b ) AF488-labeled anti-EGFR (green) to the surface (arrow) of control HT29 cells (siCL). ( c ) PEH*-Cy5.5 (red) binding is minimal. ( d – f ) The fluorescence intensities are significantly reduced in knockdown of HT29 cells (siEGFR). ( g ) Quantified results for QRH*-Cy5.5 and anti-EGFR show significantly higher intensities for siCL- vs. siEGFR-transfected cells (3.2- and 3.4-fold change, P =0.0021 and 0.0017, respectively), whereas PEH*-Cy5.5 showed a nonsignificant decrease (0.87 fold-change, P =0.57). Differences for siCL vs. siEGFR for QRH*-Cy5.5 and anti-EGFR were significantly greater than those for PEH*-Cy5.5 ( P =0.007 and 0.006, respectively). We fit two-way ANOVA models with the terms for six conditions and two replicate slides on log-transformed data. Measurements were on an average of five randomly chosen cells on two slides for each condition. ( h ) Western blot shows EGFR expression levels. ( i ) On competition, we found a significant difference in binding of QRH*-Cy5.5 to HT29 cells with the addition of unlabeled QRH* and PEH* at the concentrations of 50 μ m and higher. Nonsignficant difference was found at 0 μ m . We fit two-way ANOVA models with the terms for the labeled peptide, concentrations of the unlabeled peptides, and their interactions on log-transformed data. P -values shown here compare the difference in the intensity between unlabeled QRH* and PEH* at each dose with that at 0 μ m . Measurements are on an average of five randomly chosen cells on two slides for each condition. ANOVA, analysis of variance; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Fluorescence, Transfection, Transformation Assay, Western Blot, Expressing

    Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Characterization of EGFR peptide-binding parameters. ( a ) Apparent dissociation constant k d =50 n m , R 2 =0.95 was measured for binding of QRH*-Cy5.5 to HT29 cells. ( b ) Apparent association time constant k =0.406/min (2.46 min) was measured for binding of QRH*-Cy5.5 to HT29 cells. Both results are representative of six independent experiments.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay

    Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Peptide specific for EGFR. ( a ) Chemical structure of QRHKPRE peptide (black) with GGGSK linker (blue) and Cy5.5 fluorophore (red). ( b ) Scrambled peptide PEHKRRQ (control). ( c ) QRH*-Cy5.5 was found on the structural model to bind domain 2 of EGFR (1IVO). ( d ) Fluorescence spectra of Cy5.5-labeled peptides with λ ex =671 nm shows peak emission near 710 nm. AU, arbitrary unit; EGFR, epidermal growth factor receptor.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Fluorescence, Labeling

    Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Journal: Clinical and Translational Gastroenterology

    Article Title: EGFR Overexpressed in Colonic Neoplasia Can be Detected on Wide-Field Endoscopic Imaging

    doi: 10.1038/ctg.2015.28

    Figure Lengend Snippet: Binding of EGFR peptide and antibody to human colonic neoplasia. On confocal microscopy, binding of ( a ) QRH*-Cy5.5 peptide (red) co-localizes with that of ( b ) AF488-labeled anti-EGFR antibody (green) on surface of dysplastic colonocytes (arrow), shown in ( c ) merged image, P= 0.71. ( d ) Image contrast can be appreciated at lesion border. Magnified view of boxes in d is shown for ( e ) dysplasia and ( f ) normal. ( g ) Corresponding immunohistochemistry from a shows increased reactivity for EGFR in dysplasia. ( h ) Dysplasia ( n =29) showed significantly higher fluorescence intensities than normal ( n =15) by an average of 19.4-fold, P =1.7 × 10 −9 by two-sample t -test on log-transformed data. ( i ) Receiver operating characteristic curve shows 90% sensitivity and 93% specificity with area under curve (AUC) of 0.94 for distinguishing dysplasia from normal using peptide.

    Article Snippet: QRH*-Cy5.5 and anti-EGFR antibody showed significantly higher intensities for siCL-transfected HT29 cells than for those treated with siEGFR, whereas PEH*-Cy5.5 showed a small nonsignificant increase, .

    Techniques: Binding Assay, Confocal Microscopy, Labeling, Immunohistochemistry, Fluorescence, Transformation Assay

    Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expression in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c-Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; * p -

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Chromatin Immunoprecipitation, Western Blot, Expressing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Immunoprecipitation

    Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of the effects of modulation of EGFR, Src and Stat3 on the nuclear EGFR, Src and Stat3 complex. (A, B, and C) Immunoblotting analyses of immunecomplexes of Stat3 (IP:Stat3), EGFR (IP:EGFR), or Src (IP:Src) prepared from nuclear extracts of Panc-1 cells untransfected or transfected with Src siRNA, EGFR siRNA, or control (con) siRNA (A), or treated with or without the EGFR inhibitor (ZD1839, ZD), Src inhibitor (Dasatinib, Das), or the Stat3 inhibitor (S3I-201) for 1 or 24 h (B), or from nuclear extracts pre-incubated for 2 h with or without 100 µM pY1068, pY1086, or SPI peptide (C) and probing for EGFR, Src, Stat3; or (D) immunoblotting analysis of nuclear extracts prepared from Panc-1 cells treated or untreated with phenylarsine oxide (PAO) and probing for Src, Stat3, EGFR. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate or nuclear extract used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Transfection, Incubation

    Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells. Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Immunofluorescence with laser-scanning confocal microscopy of EGFR, Src and Stat3 association in HPDEC or Panc-1 cells. Cultured normal human pancreatic duct epithelial cells (HPDEC) (A) or pancreatic cancer, Panc-1 cells (B) were fixed, stained with primary antibodies against EGFR, Src and Stat3 and their corresponding secondary antibodies, ALexaFLuor405 (goat anti-mouse, EGFR, red), AlexaFluor488 (donkey anti-rabbit, Src, blue) and AlexaFluor546 (goat anti-rat, Stat3, green) and analyzed by laser-scanning confocal microscopy for localization (single) and colocalization (merge) studies of EGFR (red), Src (blue) and Stat3 (green) and the effects of treatment (i) without or (ii) with ZD1839 (ZD) or (iii) Dasatinib (Das) for the indicated times. Confocal images were collected using Leica TCS SP5 microscopes; Cyan, magenta, yellow and white/pale yellow arrows denote merged colors; single, one color capture, merged, three-color capture. Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunofluorescence, Confocal Microscopy, Cell Culture, Staining

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 complex in the nucleus and the sub-cellular distribution of EGFR, Src and Stat3. (A and B) Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), Stat3 (IP:Stat3), EGFR/Src (IP:EGFR/IP:Src), or of non-specific IgG non-immuneprecpitate prepared from nuclear extracts of Panc-1 or Colo-357 cells and probing for Stat3, EGFR, Src, or the Tata-binding protein (TBP); and (C), immunoblotting analysis of membrane (mem) and cytosolic (cyto) fractions and of nuclear (nuc) extracts from Panc-1 cells probing for (i) EGFR, (ii) Stat3 and (iii) Src. Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of nuclear extract used in the assay; IP:EGFR/IP:Src, sequential immunoprecipitation with anti-EGFR and then anti-Src antibody; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Binding Assay

    Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Studies of protein complex and protein binding partners using the Detection and Analysis through Nanoparticle Sizing technology. (A) Kinetic binding assay of EGFR-gold nanoparticle (GNP) probe (or mouse IgG1-GNP probe as negative control) binding to (i) EGFR protein and its complex from Panc-1 nuclear extracts, and the (ii) inhibitory effect of the mouse monoclonal anti-EGFR antibody on the EGFR-GNP probe binding to the EGFR protein; and (B) Protein complex binding partner analysis whereby the polyclonal anti-Stat3, anti-Src or anti-EGFR antibody or the non-specific rabbit IgG (negative control) is added to the assay solution prepared from the (i) non-specific mouse IgG1-GNP probe (negative control), or (ii) anti-EGFR-GNP probe; Data are representative of 4 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Protein Binding, Binding Assay, Negative Control

    Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Journal: PLoS ONE

    Article Title: A Functional Nuclear Epidermal Growth Factor Receptor, Src and Stat3 Heteromeric Complex in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0019605

    Figure Lengend Snippet: Co-immunoprecipitation with immunoblotting analysis of EGFR, Src and Stat3 association in Panc-1 and Colo-357 cells. Immunoblotting analyses of immunecomplexes of EGFR (IP:EGFR), Src (IP:Src), and Stat3 (IP:Stat3), or of non-specific IgG non-immunoprecipitate prepared from whole-cell lysates of Panc-1 or Colo-357 cells untransfected (A and B) or transfected with EGFR siRNA, Src siRNA, or control (con) siRNA (C) and probing for Src, Stat3 and EGFR in the absence (A and C) or presence (B) of Stat3 blocking peptide (Stat3 BP), Src blocking peptide (Src BP) or EGFR blocking peptide (EGFR BP). Bands corresponding to proteins in gel are shown; input: except where indicated, represents the immunoblotting for the respective immunoprecipitated protein in the same amount of lysate used in the assay; Data are representative of 3 independent studies.

    Article Snippet: Compared to anti-Stat3 or anti-Src antibody, the particle size increase upon addition of anti-EGFR antibody to the assay solution is much smaller, only slightly higher than the non-specific rabbit IgG.

    Techniques: Immunoprecipitation, Transfection, Blocking Assay