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: Oncotarget

Article Title: Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis

doi:

Figure Lengend Snippet: MDA-MB-453 or 4T1 cells were cultured with metformin (10 mM), AG825 (10 μM) or HRG-β1 (50 ng/ml) in DMEM containing 10% FBS for 24 h, then the intracellular proteins, mRNA and TCM were extracted. A. Immunoblotting for the total and phosphorylated levels (Tyr 1221/1222) of HER2 and VEGFA proteins in MDA-MB-453 cells. B. Representative images showing the mRNA and secretion levels of VEGF of MDA-MB-453 cells ( n = 5 for both). C. Representative image showing the growth curve of 4T1 tumors from control mice and those treated with metformin (200 mg/kg • day), or AG825 (10 mg/kg. day) ( n = 6 − 8). D. Immunohistochemical staining for VEGFA in 4T1 tumors; scale, 200 μm. E. Immunohistochemical staining for CD31 + vessels and quantitative measurement of microvessel density in 4T1 tumors ( n = 6 − 8). Scale bar, 75 μm. All data is presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AG825 and YC-1 were obtained from Cayman company (USA) and the recombinant human HRG-β1 was purchased from PeproTech corporation (USA).

Techniques: Cell Culture, Western Blot, Control, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Suppression of tumor angiogenesis by metformin treatment via a mechanism linked to targeting of HER2/HIF-1α/VEGF secretion axis

doi:

Figure Lengend Snippet: MDA-MB-453 cells were cultured with metformin (10 mM), AG825 (10 μM), YC-1 (10 μM) or HRG-β1 (50 ng/ml) for 24 h, then the intracellular proteins, mRNA, and tumor cell-conditioned medium (TCM) were extracted. A. YC-1 almost completely inhibited HIF-1α expression of MDA-MB-453 cells even in the presence of HRG-β1 treatment. Representative image showing mRNA levels of B. HIF-1α and C. VEGFA in MDA-MB-453 cells ( n = 5 for both). D. HUVECs were cultured with serum-free medium (SFM) or TCM from MDA-MB-453 cells treated with YC-1 (10 μM), HRG-β1 (50 ng/ml) or both ( n = 6). E. Immunoblotting for protein expression of HIF-1α in MDA-MB-453 cells in both the presence and absence of MG132 (20 μM). F. Immunohistochemical staining for HIF-1α in 4T1 tumors from control mice or those treated with metformin (200 mg/kg • day), YC-1 (10 mg/kg. day), or AG825 (10 mg/kg. day). Red arrows indicate the cells with nuclei positive for HIF-1α. Scale bar: 100 μm. G. Immunofluorescent double staining for CD31 and HIF-1α in 4T1 tumors. Scale bar: 100 μm. All data is presented as mean ± S.E.M. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: AG825 and YC-1 were obtained from Cayman company (USA) and the recombinant human HRG-β1 was purchased from PeproTech corporation (USA).

Techniques: Cell Culture, Expressing, Western Blot, Immunohistochemical staining, Staining, Control, Double Staining

Journal: Molecular Cancer

Article Title: Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism

doi: 10.1186/1476-4598-13-130

Figure Lengend Snippet: Mechanisms involved in the tumor suppression role of YY1. (a) Effects of YY1 on MUC4, ErbB2, MEF2C, MMP10 and MAPK (Erk1/2, JNK and p38) signaling pathways were assayed by western blotting. BXPC-YY1 indicates YY1overexpressing BXPC-3 cells; BXPC-Vector indicates BXPC-3 cells transfected with a control vector; BXPC-YY1 shRNA indicates YY1 knockdown BXPC-3 cells; BXPC-Scramble shRNA indicates BXPC-3 cells transfected with a vector expressing Scramble shRNA. GAPDH was used as the internal control. (b) Bands were quantified by densitometry. Histograms of the ratio (phosphorylated/constitutive form) for Erk1/2, JNK and p38 MAPK kinases are shown. (c) BXPC-YY1 shRNA cells were transfected with MMP10 siRNA or negative control siRNA. 12 h after transfection, cell migration assays were performed. The upper chambers were seeded with various cell lines. The membranes of the chambers were stained with 0.1% crystal violet. Scale bar, 100 μm. (d) Quantification of the data from Figure c. OD values from three independent experiments were assessed. (e) Luciferase activities of MMP10 promoter-transfected BXPC-Scramble shRNA or BXPC-YY1 shRNA cells treated with MUC4 siRNA or various inhibitors of signal transduction molecules. MUC4 blockage, the ErbB2 inhibitor (AG825, 25 μM) and the p38 inhibitor (SB203580, 10 μM) significantly inhibited YY1 knockdown-stimulated luciferase activity in BXPC-YY1 shRNA cells transfected with the MMP10 promoter. (f) The YY1 knockdown-stimulated luciferase activity could be significantly inhibited by MEF2C blockage. Luciferase activity of MMP10 promoter in BXPC-YY1 shRNA cells was significantly decreased when the presumed MEF2C binding site (nucleotides −881 to −890) was mutated. (*represents p < 0.05, **represents p < 0.01, # represents p < 0.001, when compared to the control group).

Article Snippet: At 12 h after transfection, the medium was replaced and cells were treated for 24 h with 25 μM AG825 (Merck Millipore), 10 μM SB203580 (Cell Signaling Technology) or equivalent amounts of DMSO vehicle (Sigma) as a control.

Techniques: Protein-Protein interactions, Western Blot, Plasmid Preparation, Transfection, Control, shRNA, Knockdown, Expressing, Negative Control, Migration, Staining, Luciferase, Transduction, Activity Assay, Binding Assay