Journal: Cell Death & Disease

Article Title: Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

doi: 10.1038/s41419-019-1882-9

Figure Lengend Snippet: a PN-1, P-EGFR, and EGFR protein levels in control MCF-7 cells, MCF-7 cells treated with EGF, and MCF-7 cells treated with EGF and AG1478 (EGFR inhibitor). b PN-1 and EGFR mRNA levels in MCF-7 cells, MDA-MB-231 cells, T47D cells and MDA-MB-468 cells. c PN-1 and EGFR protein levels in MCF-7 cells, MDA-MB-231 cells, T47D cells and MDA-MB-468 cells. d Spearman correlation analysis of the fold change of EGFR mRNA and PN-1 mRNA in human breast cancer tissues. e Spearman correlation analysis of the fold change of EGFR mRNA and PN-1 mRNA in 1104 human breast cancer tissues in starBase public database from TCGA project. f Schematic diagram shows specific inhibitors of different proteins involved in EGF/EGFR signaling pathway. g , h PN-1 mRNA(I) and protein(J) levels in control MCF-7 cells, MCF-7 cells treated with EGF, MCF-7 cells treated with EGF and inhibitors of PI3K(LY294002), PLC(U73122), JAK(Ruxolitinib), MEK(U0126) or PKC(Go6983). i PN-1 protein levels in control MCF-7 cells, MCF-7 cells treated with EGF, MCF-7 cells treated with EGF and Go6983. j Schematic diagram shows the inhibitors of different PKC isoforms. k , l PN-1 mRNA(M) and protein(N) levels in control MCF-7 cells, MCF-7 cells treated with EGF, MCF-7 cells treated with EGF and BAPT-AM (Calcium inhibitor) or rottlerin (PKCδ and PKCθ inhibitor). *** P < 0.005.

Article Snippet: Then cells were stimulated with 100 ng/mL EGF for 24 h or 1 μM PMA for 12 h. For inhibitor studies, cells were pretreated with inhibitors for 4 h before EGF or PMA stimulation, and the concentration of inhibitors were as follows according to the reported lectures: 10 μM EGFR inhibitor AG1478 (MedChemExpress, USA),10 μM PKC inhibitor Go6983 , 5 μM MEK inhibitor U0126 , 20 μM PI3K inhibitor LY294002 , 10 μM PLC inhibitor U73122 , 10 μM JAK inhibitor Ruxolitinib , 5 μM SCH772984 , 10 μM BAPTA-AM , 20 μM Rottlerin (Selleck, USA), 20 μM Mithramycin A (APExBIO, USA).

Techniques: Control

Journal: Cell Death & Disease

Article Title: Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

doi: 10.1038/s41419-019-1882-9

Figure Lengend Snippet: a PN-1 mRNA(left) and protein(right) levels in control MCF-7 cells, MCF-7 cells treated with EGF, and MCF-7 cells treated with EGF and U0126 (MEK inhibitor). b PN-1 mRNA (left) and protein (right) levels in control MCF-7 cells, MCF-7 cells treated with EGF, and MCF-7 cells treated with EGF and SCH772984 (ERK inhibitor). c , d PN-1 mRNA levels(F) and PN-1, P-ERK1/2 and ERK1/2 protein levels(G) in control MCF-7 cells, MCF-7 cells treated with PMA, and MCF-7 cells treated with PMA and U0126 or SCH772984. e PN-1 mRNA (left) and protein (right) levels in MDA-MB-231cells treated with EGF, MDA-MB-231 cells treated with EGF and AG1478, Go6983, U0126, or SCH772984. *** P < 0.005.

Article Snippet: Then cells were stimulated with 100 ng/mL EGF for 24 h or 1 μM PMA for 12 h. For inhibitor studies, cells were pretreated with inhibitors for 4 h before EGF or PMA stimulation, and the concentration of inhibitors were as follows according to the reported lectures: 10 μM EGFR inhibitor AG1478 (MedChemExpress, USA),10 μM PKC inhibitor Go6983 , 5 μM MEK inhibitor U0126 , 20 μM PI3K inhibitor LY294002 , 10 μM PLC inhibitor U73122 , 10 μM JAK inhibitor Ruxolitinib , 5 μM SCH772984 , 10 μM BAPTA-AM , 20 μM Rottlerin (Selleck, USA), 20 μM Mithramycin A (APExBIO, USA).

Techniques: Control

Journal: JCI Insight

Article Title: Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling

doi: 10.1172/jci.insight.129320

Figure Lengend Snippet: (A) G-LISA Rac1-GTP activation assay in HEKs treated with 5 μM mevastatin for 48 hours (n = 6). Treatment with 12.5 ng/mL EGF for 5 minutes served as positive control. Mevastatin induced Rac1-GTP activation. (B) ITGB5 and phalloidin staining of human keratinocytes (HEKs) treated with mevastatin or EGF in the presence or absence of 150 nM tyrphostin AG 1478. Scale bar: 10 μm. LP, lamellipodia. (C) Percentage of ITGB5+ lamellipodia+ cells (n = 3). Mevastatin induced lamellipodia formation, whereas tyrphostin AG 1478 inhibited mevastatin-induced lamellipodia. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test; ****P < 0.0001.

Article Snippet: HEKs were grown to approximately 30% confluency and switched to basal media for 24 hours and treated with 5 μM mevastatin, 150 nM tyrphostin AG 1478 (Cell Signaling Technology), or both in combination, and 12.5 ng/ml EGF treatment for 5 minutes served as a positive control.

Techniques: Activation Assay, Positive Control, Staining

Journal: Molecular cancer

Article Title: Disruption of ETV6 leads to TWIST1-dependent progression and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in prostate cancer.

doi: 10.1186/s12943-018-0785-1

Figure Lengend Snippet: Fig. 3 Epidermal growth factor receptor (EGFR) signaling triggers ETV6-mediated suppression of TWIST1. (a) Monitoring ETV6 mRNA following treatments with EGF and CI1033. PBS and DMSO, vehicle control of EGF and CI1033, respectively. (b) Monitoring TWIST1 mRNA following treatments with CI1033 and stable ETV6 expression. EV, control vector. (c) Western blot assay with cellular lysates from RasB1 cells. Cells were treated with EGFR modulators (EGF, CI1033, left panel) or a stable ETV6-expressing vector (EV vs. ETV6, right panel). (d, e) Two androgen receptor (AR)-positive cell lines transiently transfected with ETV6-specific siRNA (scr. vs. siETV6) were analyzed for TWIST1 mRNA (d) and for the Western blot assay (e). (f) Monitoring TWIST1 mRNA in 22RV1 cells transiently transfected with ETV6-specific siRNA (scr. vs. siETV6). TWIST1 mRNA was measured in response to EGFR activation (PBS vs. EGF) or inactivation (DMSO vs. CI1033). Quantification of mRNA is presented as the mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The EGF was from R&D Systems (Minneapolis, MN, USA), and the EGFR inhibitors (CI1033 and AG1478) were from Selleck (Houston, TX, USA).

Techniques: Control, Expressing, Plasmid Preparation, Western Blot, Transfection, Activation Assay

Journal: Molecular cancer

Article Title: Disruption of ETV6 leads to TWIST1-dependent progression and resistance to epidermal growth factor receptor tyrosine kinase inhibitors in prostate cancer.

doi: 10.1186/s12943-018-0785-1

Figure Lengend Snippet: Fig. 5 ETV6-TWIST1 signaling is involved in the molecular mechanism of drug resistance. (a) Proliferation assay in three DU145 derived cells treated with a tyrosine kinase inhibitor (TKI: AG1478, 0.1~ 10 μM), n = 8. shLacZ, control; shETV6, ETV6-knockdown; shETV6 + siTWIST1, both ETV6- and TWIST1-knockdown. (b) Proliferation assay in three stable RasB1 derived cells treated with a TKI (CI1033, 0.1~ 10 nM), n = 8. EV, control vector; ETV6, ETV6-expressing vector; ETV6 + TWIST1, both ETV6- and TWIST1-expressing vectors. (c) Tumor growth analysis of stable RasB1 cell lines (EV vs. ETV6) subcutaneously inoculated in male nude mice followed by treatment with CI1033. Tumor sizes were monitored weekly (left, n = 5). At the end, tumor weights were also measured (right, n = 5). (d) Selected images of mice from panel C, containing tumors (arrows) derived from stable RasB1 cell lines (EV vs. ETV6). (e) Western blot analysis of human prostate cancer cells. RasB1 cells were introduced with exogenous ETV6 or under ETV6-knockdown in 22RV1 and LNCaP cells by an siRNA approach (siETV6). EV, empty vector; scr., siRNA control. (f) Working model, activation of the epidermal growth factor receptor (EGFR) promotes tumor progression and drug resistance through RAS signaling and suppression of ETV6, which lead to TWIST1-dependent malignant phenotypes. A mutual inhibitory circuit exists between EGFR-RAS signaling and ETV6

Article Snippet: The EGF was from R&D Systems (Minneapolis, MN, USA), and the EGFR inhibitors (CI1033 and AG1478) were from Selleck (Houston, TX, USA).

Techniques: Proliferation Assay, Derivative Assay, Control, Knockdown, Plasmid Preparation, Expressing, Western Blot, Activation Assay

Journal: Toxins

Article Title: Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa

doi: 10.3390/toxins9070202

Figure Lengend Snippet: Epidermal growth factor receptor (EGFR) inhibition reduces significantly gamma-toxin-induced IL-8 production in HVECs. HVECs were exposed to HlgAB (50 µg/mL) or HlgCB (100 µg/mL) at a 1:1 molar ratio for 6 h and IL-8 production was analyzed by ELISA. IL-8 production in response to HlgAB or HlgCB was attenuated in the presence of ( A , B ) AG1478 or ( C , D ) UO126. Data show mean +/− standard deviation. Asterisks indicate significant difference from gamma-toxin treated cells with no inhibitor present ( p < 0.05).

Article Snippet: AG1478 (Tocris Bioscience, Bristol, UK, 1276) was used at a final concentration of 1 μM, TAPI (Enzo Life Sciences, Farmingdale, NY, USA, BML-PI134-0001) at 50 μM, and UO126 (Tocris Biosciences, 1144) at 10 μM.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Toxins

Article Title: Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa

doi: 10.3390/toxins9070202

Figure Lengend Snippet: IL-8 production from porcine vaginal mucosa in response to gamma-toxin involves EGFR signaling. Ex vivo PVM was used to expand on our observations in HVECs using a complex tissue model. Explants were treated topically with gamma-toxin for 6 h prior to processing for IL-8 by ELISA. Where inhibitors were used, they were applied topically 30 min prior to gamma-toxin. IL-8 produced in response to ( A ) HlgAB and ( B ) HlgCB was dose-dependent. ( C ) Gamma-toxin toxicity on PVM was not observed; ( D ) Individual subunits (HlgA, HlgB, HlgC 1000 ng/explant) did not stimulate IL-8 at equivalent doses of HlgAB or HlgCB at a 1:1 molar ratio; ( E ) AG1478 inhibition of HlgCB (500 ng/explant)-stimulated IL-8 production in PVM was dose-dependent; ( F ) HlgAB (500 ng/explant)-stimulated IL-8 production in PVM was also reduced by EGFR inhibition by addition of AG1478 (8 nmol). NC is negative (untreated) control. AG is AG1478. VC is the AG1478 vehicle control. Asterisks indicate significant difference from media alone controls (A–D) or toxin-stimulated (E,F) ( p < 0.05).

Article Snippet: AG1478 (Tocris Bioscience, Bristol, UK, 1276) was used at a final concentration of 1 μM, TAPI (Enzo Life Sciences, Farmingdale, NY, USA, BML-PI134-0001) at 50 μM, and UO126 (Tocris Biosciences, 1144) at 10 μM.

Techniques: Ex Vivo, Enzyme-linked Immunosorbent Assay, Produced, Inhibition, Control

Journal: Toxins

Article Title: Epidermal Growth Factor Receptor Signaling Enhances the Proinflammatory Effects of Staphylococcus aureus Gamma-Toxin on the Mucosa

doi: 10.3390/toxins9070202

Figure Lengend Snippet: IL-8 production from human ectocervix tissue in response to gamma-toxins. Explants were treated topically with gamma-toxin for 6 h prior to analysis of IL-8 production by ELISA. AG1478 (EGFR inhibitor) was applied topically 30 min prior to gamma-toxin. ( A ) HlgCB (1000 ng/explant) increased significantly IL-8 production from human ectocervix tissue over untreated controls; ( B ) Inhibition of EGFR signaling with AG1478 (8 nmol/explant) attenuated the IL-8 production from human ectocervical tissue in response to HlgCB (1000 ng/explant). Due to high inter-person variability in background IL-8, data are represented as fold-change in IL-8 production over unstimulated controls +/− SD. Data are normalized to fold-change and combined from 3 experiments each with n = 3. Due to physical limitations on tissue size, the effect of the vehicle control (VC) was assayed only once with n = 3. NC is negative (untreated) control. AG is AG1478. Asterisks indicate a significant difference from the vehicle control ( A ) or from toxin treatment ( B ) ( p < 0.05).

Article Snippet: AG1478 (Tocris Bioscience, Bristol, UK, 1276) was used at a final concentration of 1 μM, TAPI (Enzo Life Sciences, Farmingdale, NY, USA, BML-PI134-0001) at 50 μM, and UO126 (Tocris Biosciences, 1144) at 10 μM.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Control

Journal: bioRxiv

Article Title: HB-EGF Signaling is Required for Glucose-Induced Pancreatic β-Cell Proliferation in Rats

doi: 10.1101/683003

Figure Lengend Snippet: (A-F) Isolated rat islets were exposed to 2.8 mM glucose, 16.7 mM glucose, or HB-EGF (100 ng/ml) or betacellulin (BTC; 50 ng/ml) in the presence of 2.8 mM glucose for 72 h. (G, H) Isolated rat islets were exposed to 2.8 mM glucose and left untreated or treated with HB-EGF (100 ng/ml) with or without AG1478 (300 nM) or rapamycin (Rap; 10 nM) for 72 h. Proliferation was assessed by Ki67 (A-D, G, H) or EdU (E, F) staining and Nkx6.1 (A, B) or insulin (Ins) (C-H). Representative images of Nkx6.1 (red), Ki67 (green) and nuclei (blue) (A) or insulin (green), Ki67 or EdU (red) and nuclei (blue) (C, E, G) staining. Arrows show positive nuclei for Ki67 and EdU. (B, D, H) The percentage of Ki67+ insulin+ (or Nkx6.1+) cells of total insulin+ (or Nkx6.1+) cells. (F) Percentage of EdU+ insulin+ cells of total insulin+ cells. Data represent individual values and are expressed as mean ± SEM (n=4–6). Scale bars, 50 µm. *p < 0.05, **p<0.01, ***p<0.001, as compared to the 2.8 mM glucose control condition. ns, not significant.

Article Snippet: The EGFR tyrosine kinase inhibitor AG1478 and the mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin were from LC Laboratories (Woburn, MA).

Techniques: Isolation, Staining, Control

Journal: bioRxiv

Article Title: HB-EGF Signaling is Required for Glucose-Induced Pancreatic β-Cell Proliferation in Rats

doi: 10.1101/683003

Figure Lengend Snippet: (A,B) Isolated rat islets were exposed to 2.8 or 16.7 mM glucose with or without AG1478 (300 nM) for 72 h. (C,D) Isolated rat islets were exposed to 2.8 mM glucose, 16.7 mM glucose, or 2.8 mM glucose + 100 ng/ml HB-EGF in the absence or presence of 10 μg/ml CRM197 for 72 h. (E,F) Isolated rat islets were exposed to 2.8 mM glucose with or without 100 ng/ml HB-EGF, or 16.7 mM glucose with or without 10 μg/ml CRM197, for 72 h. (G, H) Isolated rat islets were infected with Adv-shHBEGF or Adv-shCTL and exposed to 2.8 or 16.7 mM glucose for 72 h. Proliferation was assessed by Ki67 (A-D,G,H) or pH3 (E,F) staining and insulin (Ins; A-D,G,H) or Nkx6.1 (E,F) staining to mark β-cells. (A, C, E, G) Representative images of insulin (green) or Nkx6.1 (red), Ki67 (red) or pH3 (green) and nuclei (blue) staining. Arrows show positive nuclei for Ki67 or pH3. (B, D, H) Percentage of Ki67+ insulin+ cells of total insulin+ cells. (F) Percentage of pH3+ Nkx6.1+ cells of total Nkx6.1+ cells. Data represent individual values and means ± SEM (n=4– 6). Scale bars, 50 µm. **p<0.01, ***p<0.001, ****p<0.0001, as compared to the 2.8 mM glucose condition. && p<0.01, as compared to 16.7 mM glucose. ns, not significant.

Article Snippet: The EGFR tyrosine kinase inhibitor AG1478 and the mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin were from LC Laboratories (Woburn, MA).

Techniques: Isolation, Infection, Staining

Journal: Oncotarget

Article Title: Multi-factorial modulation of colorectal carcinoma cells motility – partial coordination by the tetraspanin Co-029/tspan8

doi: 10.18632/oncotarget.16247

Figure Lengend Snippet: (A) Increased motility of Is1-Co029 cells induced by chemical inhibitor of EGFR, AG1478, (B) by silencing of EGFR, and reversion by Co-029 silencing or addition of the Co-029 mAb Ts29.1 (for si-CTRL = siCD53). (C) Increased motility induced by Cetuximab and reversion by addition of the Co-029 mAb Ts29.1. (D) Inhibition of Is1-Co029 cell motility by integrins VLA1 and VLA2 RNAi. Asterisks indicates p≤.0001.

Article Snippet: In addition to various siRNA, Cetuximab (25 μg/ml), the EGFR inhibitor AG1478 (Merck, Germany) at 5μM, PD98059 for Mek1 (20μM), U0126 (Promega) at 10μM for the MAPK Erk, SB203580 (Calbiochem) at 20 nM for the MAPK p38, D-JNKI-1 targeting JNK1 (a gift of Xigen SA) at 3μM were used in motility experiments as well as the TLR2 ligand B. subtilis peptidoglycan, PGN (InvivoGen, San Diego).

Techniques: Inhibition

Journal: Molecular Vision

Article Title: Osmotic regulation of aquaporin-8 expression in retinal pigment epithelial cells in vitro: Dependence on K ATP channel activation

doi:

Figure Lengend Snippet: Receptor-mediated regulation of NaCl-induced expression of the AQP8 gene in RPE cells. The level of AQP8 mRNA was determined with real-time RT–PCR analysis in cells cultured for 6 h in iso- (control) and hyperosmotic (+ 100 mM NaCl) media (as indicated by the panels of the bars), and is expressed as folds of the unstimulated control. A : The following agents were tested: the inhibitor of the FGF receptor kinase, PD173074 (500 nM), the blocker of VEGF receptor-2, SU1498 (10 µM), the inhibitor of the PDGF receptor tyrosine kinase, AG1296 (10 µM), the blocker of the EGF receptor tyrosine kinase, AG1478 (600 nM), the inhibitor of TGF-β1 superfamily activin receptor-like kinase receptors, SB431542 (10 µM), the inhibitor of various G protein-coupled receptor subtypes, suramin (200 µM), the broad-spectrum matrix metalloproteinase inhibitor 1,10-phenanthroline (1,10-Phen; 10 µM), a recombinant human IL-1 receptor antagonist (IL-1RA; 1 µg/ml), and the NLRP3 inflammasome inhibitor‎ MCC950 (1 µM). B : Effect of exogenous IL-1β (10 ng/ml) on AQP8 gene expression. Vehicle control was made with dimethyl sulfoxide (DMSO; 1:500). Each bar represents mean ± standard error of the mean (SEM) obtained in three to ten independent experiments using cell lines from different donors. Significant difference vs. unstimulated control: *p<0.05. Significant difference vs. NaCl control: ● p<0.05.

Article Snippet: Ac-YVAD-CMK, AG1478, and Stattic were from Enzo Life Science (Lausen, Switzerland).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Recombinant