Journal: Science Advances

Article Title: Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

doi: 10.1126/sciadv.1600844

Figure Lengend Snippet: ( A to C ) Dual immunofluorescence detection and quantification of cTnT + /Ki67 + proliferating cardiomyocytes at 3 days after MI at the mid-infarct level of mouse left ventricles. Arrows indicate doubly positive cells. ( D to F ) Dual immunofluorescence detection and quantification of ErbB2 + /cTnT + cardiomyocytes at 3 days after MI at the mid-infarct level suggest the involvement of NRG1 signaling in zECM-treated groups. All image analyses were performed using 20 × 10–μm areas in five images of each heart ( n = 4 per group). All quantitative data represent means ± SD. *** P < 0.001 compared to mECM and saline; # P < 0.05 for hzECM versus nzECM. Scale bars, 50 μm.

Article Snippet: For ErbB2 inhibition in vivo, the ErbB2 inhibitor AG825 (sc-202045A, Santa Cruz Biotechnology) dissolved in DMSO was intraperitoneally injected once at 5 mg/kg immediately after the cardiac ECM administration ( , ).

Techniques: Immunofluorescence, Saline

Journal: Science Advances

Article Title: Decellularized zebrafish cardiac extracellular matrix induces mammalian heart regeneration

doi: 10.1126/sciadv.1600844

Figure Lengend Snippet: To inhibit ErbB2 activity in vivo, the ErbB2 inhibitor AG825 was intraperitoneally injected once (5 mg/kg) immediately after the administration of decellularized cardiac ECM. Cardiac contractile function is indicated by ( A ) fractional area change and ( B ) ejection fraction; LV dimension is indicated by ( C ) EDA and ( D ) ESA. No significant difference is observed between all groups at all time points ( n = 7 per group; all P > 0.05; data analyzed by two-way repeated-measures ANOVA). Dual immunofluorescence detection and quantification of ( E and F ) c-kit + /Ki67 + proliferating cardiac stem cells and ( G and H ) ErbB2 + /cTnT + cardiomyocytes. No significant difference is observed between all groups ( n = 4 per group, all P > 0.05). Scale bars, 50 μm.

Article Snippet: For ErbB2 inhibition in vivo, the ErbB2 inhibitor AG825 (sc-202045A, Santa Cruz Biotechnology) dissolved in DMSO was intraperitoneally injected once at 5 mg/kg immediately after the cardiac ECM administration ( , ).

Techniques: Activity Assay, In Vivo, Injection, Immunofluorescence

Journal: Stem Cells and Development

Article Title: A Paracrine Mechanism Accelerating Expansion of Human Induced Pluripotent Stem Cell-Derived Hepatic Progenitor-Like Cells

doi: 10.1089/scd.2014.0479

Figure Lengend Snippet: Effect of erbB receptors and Fn14 signaling in human iPS cell-derived HPCs. (A) Histograms of erbB family expression (EGFR, CD340/erbB2, erbB3, and erbB4) on P0 CD13+CD133+ HPCs derived from human iPS cells. white, isotype control; gray, stained with the antibody against the cell surface molecule. Expression of the isotype control set the gate in approximately <1%. Representative data are shown (two independent experiments). (B) Effect of EGF and NRG1 on the expansion of human iPS cell-derived HPCs. P0 CD13+CD133+ HPCs were incubated without EGF, with NRG1 only, EGF only (standard culture medium), or EGF plus NRG1. (C) Effect of a specific inhibitor of erbB2 on the expansion of human iPS cell-derived HPCs. P0 CD13+CD133+ HPCs were incubated with AG825. (B, C) The panels show the numbers of large colonies (consisting of over 200 cells) derived from 1,500 sorted cells. Results are represented as the mean colony count±SD (triplicate culture samples). *P<0.05. Representative data are shown (two independent experiments). (D) Expression levels of EGF, NRG1, and TWEAK were measured in MEFs (after 0, 2, and 6 days of culture), NIH3T3 cells, and mouse E12 whole embryo (E12WE) cells by quantitative real-time PCR analyses. Gene expression was normalized to the expression of a housekeeping gene (HPRT1). The expression of genes in WE12 whole embryo cells was set to 1.0. Values are the mean±SD from two independent experimental samples. ND indicates not detected. (E) Effect of an Fn14 blocking antibody on the expansion of human iPS cell-derived HPCs. P0 CD13+CD133+ HPCs were cultured with an anti-human Fn14 blocking antibody. (F) Effect of an Fn14 ligand on expansion of human iPS cell-derived HPCs. P0 CD13+CD133+ HPCs were cultured with recombinant human TWEAK. (E, F) The panels show the numbers of large colonies (consisting of over 200 cells) derived from 1,500 sorted cells. Results are represented as the mean colony count±SD (triplicate culture samples). **P<0.01. Representative data are shown (two independent experiments). EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; NRG1, neuregulin 1; TWEAK, tumor necrosis factor-like weak inducer of apoptosis.

Article Snippet: To block the effect of erbB receptor signaling, cells were incubated with the erbB2 inhibitor, AG825 (5 μM; Cayman Chemical, Ann Arbor, MI), 1 day after plating.

Techniques: Derivative Assay, Expressing, Control, Staining, Incubation, Real-time Polymerase Chain Reaction, Gene Expression, Blocking Assay, Cell Culture, Recombinant

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Transactivated Epidermal Growth Factor Receptor Recruitment of α-actinin-4 From F-actin Contributes to Invasion of Brain Microvascular Endothelial Cells by Meningitic Escherichia coli

doi: 10.3389/fcimb.2018.00448

Figure Lengend Snippet: Meningitic E. coli PCN033-induced activation of EGFR contributes to its invasion of hBMECs. (A) Effects of the EGFR inhibitor gefitinib, AG1478 and the analog AG825 on PCN033 invasion of hBMECs. Data represent the mean ± SD of three duplications and are presented here as relative invasion compared with the vehicle control. (B) Verification of successful EGFR KO and its influence on bacterial invasion. (C) Tyrosine phosphorylation of EGFR during the infection. (D) EGFR activation in response to both viable PCN033 and heat-inactivated PCN033 strains. (E) Quantitation analysis of different EGFR ligands in response to PCN033 infection. GAPDH was used as the endogenous control. (F) Comparison of the induction of HB-EGF by the viable and inactivated PCN033 over time. (G) Effect of CRM197 treatment on the PCN033-induced EGFR activation. hBMECs were challenged with PCN033 for 3 h. (H) The effect of CRM197 on PCN033 invasion of hBMECs. The results represent the relative invasion percentage compared with the untreated control. * p < 0.05, ** p < 0.01.

Article Snippet: The EGFR tyrosine kinase inhibitor AG1478 and Gefitinib, the ErbB2 inhibitor AG825, the ADAM17 selective inhibitor TAPI-1 were purchased from Medchem express (Princeton, NJ, USA), and the HB-EGF inhibitor CRM197 was purchased from Pfenex Inc. (San Diego, CA, USA).

Techniques: Activation Assay, Infection, Quantitation Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Transactivated Epidermal Growth Factor Receptor Recruitment of α-actinin-4 From F-actin Contributes to Invasion of Brain Microvascular Endothelial Cells by Meningitic Escherichia coli

doi: 10.3389/fcimb.2018.00448

Figure Lengend Snippet: Schematic representation of the mechanism involving EGFR in meningitic bacterial penetration of the BBB. Bacterial infection of hBMECs induces the ligand (HB-EGF)-dependent transactivation of EGFR, which requires the cleavage activity of ADAM17. Activated EGFR dimerizes with its heterogenous partner ErbB3, and then competitively recruits and disassembles ACTN4 from the intracellular F-actin fibers, leading to breakdown as well as reorganization of the actin cytoskeleton, which eventually facilitates bacterial invasion.

Article Snippet: The EGFR tyrosine kinase inhibitor AG1478 and Gefitinib, the ErbB2 inhibitor AG825, the ADAM17 selective inhibitor TAPI-1 were purchased from Medchem express (Princeton, NJ, USA), and the HB-EGF inhibitor CRM197 was purchased from Pfenex Inc. (San Diego, CA, USA).

Techniques: Infection, Activity Assay

Journal: Oncogene

Article Title: LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression

doi: 10.1038/onc.2009.221

Figure Lengend Snippet: ErbB2 and PI3K activity are necessary for LMO4 expression. ( a ) Western blot analysis of LMO4 expression in BT-474 cells treated with the following small molecule inhibitors: ZD1836 (EGFR, 1 µM), AG825 (ErbB2, 20 µM), LY294002 (PI3K, 40 µM), PD98059 (MEK, 20 µM), and JNK Inhibitor II (JNK, 25 µM). β-actin was used as a loading control. ( b ) Quantification of (a). LMO4 was expressed relative to β-actin. Values are the average of at least 4 independent experiments. Error bars depict standard deviations. * p<0.05 and ** p<5×10 −4 compared to vehicle. ( c ) Total RNA was isolated 12 hrs after treatment of BT-474 cells with AG825 (ErbB2, 20 µM) and analyzed for LMO4 mRNA expression using real time RT-PCR. Values represent the averages of at least 4 experiments. * p<5.0 × 10 −7 compared to vehicle-treated cells. ( d ) (left panel) BT-474 cells were treated with vehicle or AKT Inhibitor V (AKT, 5 µM) for 24 hrs and then cell lysates were collected for immunoblotting against LMO4. phospho-AKT (pAKT) was used to demonstrate inhibitor efficacy while total AKT and β-actin were used as loading controls. (right panel) HEK-293T cells were transiently transfected with 1.5 µg of myristolated AKT (myr-AKT), kinase inactive AKT (K179M AKT) or empty vector. Cells were lysed after 48 hrs and analyzed for LMO4 protein expression. p-AKT and total AKT levels are shown to confirm plasmid activity. β-actin was used as loading control. ( e ) (upper panel) Schematic of the LMO4 gene which has 5 exons (E1-E5) and two promoters (P1 and P2). The alternative transcription start sites yield identical protein products because the translation start site (ATG) is in Exon 2. The 5’ and 3’ ends of each construct are indicated below each promoter. (bottom panel) Activity from two proximal LMO4 promoter constructs (P1) and one distal LMO4 promoter construct (P2) was assessed in BT-474 cells treated with various inhibitors. All luciferase values were first normalized to a renilla transfection efficiency control and then expressed relative to values obtained with a minimal SV40 promoter. * p<5×10 −4 , ˆ p<0.05, ˆˆ p<0.001.

Article Snippet: Following 24 hrs of serum-starvation, heregulin (377-HB, 5 ng/mL, R&D systems, Minneapolis, MN, USA) or the following inhibitors were used: Tyrphostin AG825 (ErbB2, 20 μM, Calbiochem, San Diego, CA, USA), ZD1836 (EGFR, 1 μM, AstraZeneca, Wilmington, DE, USA), LY294002 (PI3K, 40 μM, Promega, Madison, WI, USA), PD98059 (MEK, 20 μM, Promega), JNK Inhibitor II (JNK, 25 μM, Calbiochem) and AKT Inhibitor V (AKT, 5 μM, Calbiochem).

Techniques: Activity Assay, Expressing, Western Blot, Control, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Luciferase

Journal: Oncogene

Article Title: LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression

doi: 10.1038/onc.2009.221

Figure Lengend Snippet: ErbB2 and PI3K activity are necessary for LMO4 expression. ( a ) Western blot analysis of LMO4 expression in BT-474 cells treated with the following small molecule inhibitors: ZD1836 (EGFR, 1 µM), AG825 (ErbB2, 20 µM), LY294002 (PI3K, 40 µM), PD98059 (MEK, 20 µM), and JNK Inhibitor II (JNK, 25 µM). β-actin was used as a loading control. ( b ) Quantification of (a). LMO4 was expressed relative to β-actin. Values are the average of at least 4 independent experiments. Error bars depict standard deviations. * p<0.05 and ** p<5×10 −4 compared to vehicle. ( c ) Total RNA was isolated 12 hrs after treatment of BT-474 cells with AG825 (ErbB2, 20 µM) and analyzed for LMO4 mRNA expression using real time RT-PCR. Values represent the averages of at least 4 experiments. * p<5.0 × 10 −7 compared to vehicle-treated cells. ( d ) (left panel) BT-474 cells were treated with vehicle or AKT Inhibitor V (AKT, 5 µM) for 24 hrs and then cell lysates were collected for immunoblotting against LMO4. phospho-AKT (pAKT) was used to demonstrate inhibitor efficacy while total AKT and β-actin were used as loading controls. (right panel) HEK-293T cells were transiently transfected with 1.5 µg of myristolated AKT (myr-AKT), kinase inactive AKT (K179M AKT) or empty vector. Cells were lysed after 48 hrs and analyzed for LMO4 protein expression. p-AKT and total AKT levels are shown to confirm plasmid activity. β-actin was used as loading control. ( e ) (upper panel) Schematic of the LMO4 gene which has 5 exons (E1-E5) and two promoters (P1 and P2). The alternative transcription start sites yield identical protein products because the translation start site (ATG) is in Exon 2. The 5’ and 3’ ends of each construct are indicated below each promoter. (bottom panel) Activity from two proximal LMO4 promoter constructs (P1) and one distal LMO4 promoter construct (P2) was assessed in BT-474 cells treated with various inhibitors. All luciferase values were first normalized to a renilla transfection efficiency control and then expressed relative to values obtained with a minimal SV40 promoter. * p<5×10 −4 , ˆ p<0.05, ˆˆ p<0.001.

Article Snippet: Following 24 hrs of serum-starvation, heregulin (377-HB, 5 ng/mL, R&D systems, Minneapolis, MN, USA) or the following inhibitors were used: Tyrphostin AG825 (ErbB2, 20 μM, Calbiochem, San Diego, CA, USA), ZD1836 (EGFR, 1 μM, AstraZeneca, Wilmington, DE, USA), LY294002 (PI3K, 40 μM, Promega, Madison, WI, USA), PD98059 (MEK, 20 μM, Promega), JNK Inhibitor II (JNK, 25 μM, Calbiochem) and AKT Inhibitor V (AKT, 5 μM, Calbiochem).

Techniques: Activity Assay, Expressing, Western Blot, Control, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Luciferase

Journal: Oncogene

Article Title: LMO4 is an essential mediator of ErbB2/HER2/Neu-induced breast cancer cell cycle progression

doi: 10.1038/onc.2009.221

Figure Lengend Snippet: ErbB2 and PI3K activity are necessary for LMO4 expression. ( a ) Western blot analysis of LMO4 expression in BT-474 cells treated with the following small molecule inhibitors: ZD1836 (EGFR, 1 µM), AG825 (ErbB2, 20 µM), LY294002 (PI3K, 40 µM), PD98059 (MEK, 20 µM), and JNK Inhibitor II (JNK, 25 µM). β-actin was used as a loading control. ( b ) Quantification of (a). LMO4 was expressed relative to β-actin. Values are the average of at least 4 independent experiments. Error bars depict standard deviations. * p<0.05 and ** p<5×10 −4 compared to vehicle. ( c ) Total RNA was isolated 12 hrs after treatment of BT-474 cells with AG825 (ErbB2, 20 µM) and analyzed for LMO4 mRNA expression using real time RT-PCR. Values represent the averages of at least 4 experiments. * p<5.0 × 10 −7 compared to vehicle-treated cells. ( d ) (left panel) BT-474 cells were treated with vehicle or AKT Inhibitor V (AKT, 5 µM) for 24 hrs and then cell lysates were collected for immunoblotting against LMO4. phospho-AKT (pAKT) was used to demonstrate inhibitor efficacy while total AKT and β-actin were used as loading controls. (right panel) HEK-293T cells were transiently transfected with 1.5 µg of myristolated AKT (myr-AKT), kinase inactive AKT (K179M AKT) or empty vector. Cells were lysed after 48 hrs and analyzed for LMO4 protein expression. p-AKT and total AKT levels are shown to confirm plasmid activity. β-actin was used as loading control. ( e ) (upper panel) Schematic of the LMO4 gene which has 5 exons (E1-E5) and two promoters (P1 and P2). The alternative transcription start sites yield identical protein products because the translation start site (ATG) is in Exon 2. The 5’ and 3’ ends of each construct are indicated below each promoter. (bottom panel) Activity from two proximal LMO4 promoter constructs (P1) and one distal LMO4 promoter construct (P2) was assessed in BT-474 cells treated with various inhibitors. All luciferase values were first normalized to a renilla transfection efficiency control and then expressed relative to values obtained with a minimal SV40 promoter. * p<5×10 −4 , ˆ p<0.05, ˆˆ p<0.001.

Article Snippet: Following 24 hrs of serum-starvation, heregulin (377-HB, 5 ng/mL, R&D systems, Minneapolis, MN, USA) or the following inhibitors were used: Tyrphostin AG825 (ErbB2, 20 μM, Calbiochem, San Diego, CA, USA), ZD1836 (EGFR, 1 μM, AstraZeneca, Wilmington, DE, USA), LY294002 (PI3K, 40 μM, Promega, Madison, WI, USA), PD98059 (MEK, 20 μM, Promega), JNK Inhibitor II (JNK, 25 μM, Calbiochem) and AKT Inhibitor V (AKT, 5 μM, Calbiochem).

Techniques: Activity Assay, Expressing, Western Blot, Control, Isolation, Quantitative RT-PCR, Transfection, Plasmid Preparation, Construct, Luciferase