Journal: The Journal of Biological Chemistry

Article Title: Metformin Activates AMP-activated Protein Kinase by Promoting Formation of the αβγ Heterotrimeric Complex *

doi: 10.1074/jbc.M114.604421

Figure Lengend Snippet: Metformin promoted the formation of the AMPKαβγ heterotrimeric complex in hepatocytes. a, cell lysates from untreated Hepa1–6 cells (left panel) and Hepa1–6 cells treated with metformin (0.5 mm, 5 h) (right panel) were incubated with normal IgG and antibodies against β1 and 2 together with protein G beads (16 h, 4 °C). Supernatant and magnetic beads (pellet) were collected separately. b and c, Hepa1–6 cells treated with metformin (0.5 mm. 5h) were harvested in Cell Lysis buffer (Cell Signaling) (b) or RIPA buffer (c) and incubated with antibodies against β1/2 (Cell Signaling). Immunoprecipitates were washed and immunoblotted with the indicated antibodies. d, metformin treatment (50 mg/kg, 2 weeks) increased the association of endogenous α subunit with endogenous β and γ subunits in the liver of mice fed a high-fat diet (each lane represents an individual mouse sample).

Article Snippet: AMPKα1, β1, and γ1 were immunoprecipitated using β1/2 antibodies (Cell Signaling).

Techniques: Incubation, Magnetic Beads, Lysis

Journal: Molecules

Article Title: The Effect of DA-9701 on 5-Hydroxytryptamine-Induced Contraction of Feline Esophageal Smooth Muscle Cells

doi: 10.3390/molecules19045135

Figure Lengend Snippet: PLC β1 mediated the 5-HT-induced contraction of permeabilized ESMCs . Data are expressed as the means ± S.E. of four experiments (Student’s t -test; ** p < 0.01 vs. control).

Article Snippet: 2-methyl-5-hydroxytryptamine HCl (Tocris, Bristol, UK), DA-9701 (Dong-A Pharmaceutical Co., Seoul, Korea ), G protein antibodies (G i1 , G i2 , G i3 , G q , G s , G o , and G β), PLC isozyme antibodies (β1, β3, γ1), phosphospecific Ser 19 -MLC 20 antibody, and GAPDH antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Ser 19 -MLC 20 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques:

Journal: Journal of materials chemistry. B

Article Title: Surface modification of polymeric electrospun scaffolds via a potent and high-affinity integrin α4β1 ligand improved adhesion, spreading and survival of human chorionic villus-derived mesenchymal stem cells: a new sight for fetal tissue engineering

doi: 10.1039/c9tb02309g

Figure Lengend Snippet: Binding mechanism of LLP2A to CV-MSCs. (A) Integrin α4 and β1 were highly expressed on CV-MSCs. (B) LLP2A effectively bound to CV-MSCs (blue curve), and the binding efficiency was markedly blocked by anti-integrin α4 and anti-integrin β1 antibodies together (orange curve), anti-integrin β1 antibody only (green curve), or anti-integrin α4 antibody only (black curve).

Article Snippet: To test the expression of the α4 and β1 integrin subunits on CV-MSCs and HCAECs, samples were stained with mouse anti-human α4 or β1 integrin antibodies (Millipore) on ice for 30 min, washed three times with wash buffer, incubated with goat anti-mouse 546 conjugate (Life Technologies) in DPBS on ice for 30 min, and then washed with DPBS.

Techniques: Binding Assay

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: Design and characterization of PCB/INS particles. (A) The strategy for construction of PCB/INS particles/capsule. (B) The loading efficiency and (C) loading content of insulin for different PCB/INS particles at mass ratio of 8:1 and 10:1 between PCB and insulin, respectively. (D) TEM images of particles. The particles were negatively stained with phosphotungstic acid. The scale bar was 500 nm, 2 µm and 200 nm for PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles image, respectively. (E) The 3D-SIM images of the PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles with FITC-labeled insulin. The white scale bar: 5 µm. The yellow scale bar in insert images: 1 µm. The mean ± SD was shown (n = 3). n.s. > 0.05, * P < 0.05.

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Staining, Labeling

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: The release and activity of insulin. (A) The zeta potential of particles at different pH microenvironments. (B) Release profiles of insulin from particles at distinct pH microenvironment. (C) TEM images of PCB/INS particles in phosphate buffer saline (PBS) (pH 7.4) after 24 h incubation. The particles were negatively stained with phosphotungstic acid. The white arrows indicated the fragments. The scale bar was 500 nm, 1 µm and 200 nm for PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles image, respectively. (D) CD spectra of the native insulin and insulin released from the particles. (E) The percentage of insulin undergraded at different time points in the trypsin solution. (F) Representative motion trajectories of the PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles with FITC-labeled insulin in mucus at a time lapse of 7 s. The mean ± SD was shown (n = 3).

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Activity Assay, Zeta Potential Analyzer, Saline, Incubation, Staining, Circular Dichroism, Labeling

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: The CLDN4 protein and mRNA level of Caco-2 cell monolayers after treatment. (A) Western blot results of CLDN4 after 2 h of PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles incubation at a concentration of 5 µg PCB/mL or removing. (B) Western bands were scanned and normalized to the gray values of internal control β-actin. Quantified results of CLDN4 in (A) using ImageJ. The data was relative to the untreated control in each Western band. The untreated control was settled to 1. (C) Fluorescence images collected by CLSM after 2 h of PCB 122 /INS particles treatment or removal. CLDN4 was shown in green, and cell nuclei stained with DAPI were in blue. (D) Relative CLDN4 mRNA level in Caco-2 cells after 2 h of PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles incubation or removal. The mean ± SD was shown (n = 3). * P < 0.05, ** P < 0.01.

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Western Blot, Incubation, Concentration Assay, Control, Fluorescence, Staining

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: The mechanism for PCB/INS particles opening the tight junctions. (A) Western blot results of CLDN4 expression after 2 h of PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles treatment in the presence of bafilomycin A1. Western bands were scanned and normalized to the gray values of internal control β-actin. The results were quantified using ImageJ. (B) Western blot results of CLDN4 expression after 2 h of PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles treatment in the presence of various endocytic inhibitors. (C) Fluorescence images of Caco-2 cell monolayers after incubation with PCB 122 /INS particles for 2 h. CLDN4 was shown in green. Endosomes/lysosomes stained with LysoTracker deep red were in red. Cell nuclei stained with DAPI were in blue. (D) Fluorescence images collected by CLSM. F-actin stained with FITC-labeled phalloidin was shown in green, and the cell nuclei stained with DAPI were in blue. (E) Western blot results of CLDN4 expression after 2 h of PCB 23 /INS, PCB 36 /INS and PCB 122 /INS particles treatment in the presence of integrin αV antibody or integrin β1 antibody. (F) Western bands were scanned and normalized to the gray values of internal control β-actin. Quantified results of CLDN4 in (E) using ImageJ. (G) The schematic diagram for the mechanism. For Western blot, the data was relative to the untreated control in each Western band. The untreated control was settled to 1. The mean ± SD was shown (n = 3).

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Western Blot, Expressing, Control, Fluorescence, Incubation, Staining, Labeling

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: PCB 122 /INS particles enabled oral insulin delivery in STZ-induced type 1 diabetic rats. (A) Variation of blood glucose levels of diabetic rats after treatment for 12 h. (B) Variation of serum insulin level of diabetic rats after treatment for 12 h. Diabetic rats were orally administered PCB/INS particles, insulin solution at a dose of 75 IU/kg, PCB/INS particles in capsules at a dose of 50 IU/kg. Subcutaneous (SC) injection of insulin solution at a dose of 5 IU/kg, or saline via gavage. (C) Variation of blood glucose levels of diabetic rats after treatment. (D) Variation of serum insulin level of diabetic rats after treatment. Diabetic rats were orally administered PCB 122 /INS particles at a dose of 75 IU/kg and PCB 122 /INS particles in capsules at a dose of 50 IU/kg once a day for five consecutive days. (E) In vivo glucose tolerance test toward diabetic mice 4 h post-administration of PCB 122 /INS particles at a dose of 75 IU/kg or PCB 122 /INS particles in capsules at a dose of 50 IU/kg. The mean ± SD was shown (n = 5).

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Capsules, Injection, Saline, In Vivo

Journal: Theranostics

Article Title: Charge-switchable zwitterionic polycarboxybetaine particle as an intestinal permeation enhancer for efficient oral insulin delivery

doi: 10.7150/thno.54176

Figure Lengend Snippet: The biocompatibility and safety of PCB 122 /INS particles. (A) Western blot results of CLDN4 in small intestine of diabetic rats treated with PCB 122 /INS particles for 24 h. Western bands were scanned and normalized to the gray values of internal control GAPDH. The results were quantified using ImageJ. The data was relative to the untreated control in each Western band. The untreated control was settled to 1. The mean ± SD was shown (n = 3). (B) Fluorescence images of small intestines collected by CLSM. CLDN4 was shown in red, and cell nuclei stained with DAPI were in blue. The scale bar was 50 µm. (C-F) The endotoxin and pro-inflammatory cytokines in blood serum that was collected 24 h after the last administration. (G) Hematoxylin-eosin staining of small intestines. The scale bar was 100 µm. Diabetic rats were orally administered PCB 122 /INS particles at a dose of 75 IU/kg and PCB 122 /INS particles in capsules at a dose of 50 IU/kg once a day for five consecutive days. The mean ± SD was shown (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-integrin αV and β1 antibodies were purchased from Affinity Biosciences LTD. All plates used in cellular experiments were Corning Incorporated.

Techniques: Western Blot, Control, Fluorescence, Staining, Capsules

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Optimal intensity of ESW (10 kV for 500 impulses) accelerated osteoblast adhesion. Data are presented as the mean ± S.D. (error bars) in triplicate independent experiments (n = 3). The data show that at the times of 2, 4, 6, 8, and 10 h after ESW treatment, the number of adhesive osteoblasts was significant higher than the number without ESW treatment. p < 0.01 as compared with the control group at the same period. When the siItgb1 was added prior to ESW treatment, the promotion of adhesion of osteoblasts by ESW was inhibited. p < 0.01 as compared with the ESW group at the same period. p > 0.05 as compared with the control group at the same period. It was observed that siItga5 also inhibited the ESW-induced adhesion although not as significantly as did siItgb1. The promotion of adhesion induced by ESW was abrogated, whereas integrin α5 and β1 subunits were silenced. p < 0.01 as compared with the ESW group at the same period.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Adhesive, Control

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: A–D, ESW-induced elevations of mRNA level of α5 and β1 integrin of osteoblasts, peaking at 1 h (B). The specific inhibitors for signal transduction pathways had no influence on the integrin expression (C). A and C, representative electrophoretic images. The osteoblasts were harvested to extract total RNA 0.5, 1, 2, 4, 8, and 12 h after 500 impulses of 10-kV shock wave treatment. The cells without ESWT were run as control groups. After standardization of housekeeping gene expression, equal amounts of cDNA from each sample were subjected to 36 cycles to amplify Itga5 and Itgb1 mRNA expression. The values of the control group were normalized to 100%. a, p > 0.05; b, p < 0.01; c, p < 0.05 as compared with the control group at certain time periods. In addition, several signal transduction pathway inhibitors were added to the samples for 1 h prior to ESWT. 2 h after ESWT, the samples were collected to extract RNA and to analyze whether the Itga5 and Itgb1 mRNA were influenced by signal pathway inhibitors listed above. Our data showed that no influence on the expression of Itga5 or Itgb1 mRNA was observed under the conditions with or without inhibitors (p > 0.05). Error bars, S.D.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Transduction, Expressing, Control, Gene Expression

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: ESW enhanced integrin α5 and β1 subunit protein production in 2 h according to the data from flow cytometry analysis and Western blotting. For flow cytometry, osteoblasts from experimental groups and the control group were stained with PE-conjugated anti-rat Itga5 or Itgb1 antibody under the guidance of the manufacturer. It is shown that both Itga5 and Itgb1 proteins increased significantly in 2 h in the experimental group (A). *, p = 0.021; #, p < 0.01 as compared with the blank control group. Samples with or without ESWT and those associated with siItga5 and/or siItgb1 prior to ESW were subjected to radioimmune precipitation assay lysis. Western blotting was applied to analyze whether Itga5 and Itgb1 expression levels changed in the protein extractions. The same results are shown as those indicated by flow cytometry (B and C). In addition, the data from the ESW plus siRNA groups indicated that the siRNA reagents for both Itga5 and Itgb1 were effective (B and C). a, p < 0.05; b, p < 0.01 as compared with the blank control group. #, p < 0.01 as compared with ESW group. Results are presented with mean values ± S.E. (error bars) calculated from four paired triplicate experiments.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Flow Cytometry, Western Blot, Control, Staining, Lysis, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Evaluations of phosphorylation levels of focal adhesion kinase surrounding Tyr-397, Tyr-576/577, and Tyr-925. After experimental groups were subjected to direct exposure to 10 kV for 500 impulses of ESWT, we collected the extracts at 0.5, 1, 2, 4, or 8 h. Samples without ESWT were set as the control group. Then the extracts were quantified in triplicate using Western blotting and normalized by β-actin expression (A). A marked elevation of FAK phosphorylation (p-FAK) at Tyr-397 peaking at 4 h was observed, and we also observed a slight increase of FAK phosphorylation at Tyr-925 in 2 h after ESWT. ESW had no influence on the expression of FAK phosphorylation at Tyr-576/577 (Fig. 5A). A representative electrophoretic image of the study on FAK phosphorylation at Tyr-397 influenced by siRNAs of integrins is also depicted (B). 4 h after the optimal dose of ESWT, a decline in the expression of phosphorylated FAK at Tyr-397 was observed in the group with siRNA pretreatment. However, total protein expression levels of FAK were not affected by silencing of Itga5 and/or Itgb1 (Fig. 5B). Moreover, several specific cell signal pathway inhibitors, namely PD98059, U0126, LY294002, SB203580, SP600125, H-89, and AG490, were also added to the osteoblasts for 1 h, respectively, before ESWT. Both bands of total FAK and β-actin showed equal amounts of proteins subjected to protein electrophoresis (C). The data indicated that both PD98059 and U0126, unlike other inhibitors listed, inhibited ESW-induced FAK phosphorylation at Tyr-397 (D). Data represent the mean ± S.E. (error bars) in triplicate independent experiments (n = 3). The values of the control group were normalized to 100%. a, p > 0.05; b, p < 0.05; c, p < 0.01 as compared with the control group at the same time. d, p < 0.01; e, p > 0.05 versus ESW group.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Phospho-proteomics, Control, Western Blot, Expressing, Protein Electrophoresis

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Enhancement of β-catenin activity by ESW (500 impulses at 10 kV) stimulation after elevation of integrin α5 and β1 expression. ESW raised β-catenin phosphorylation in 3 h (A). ERK1/2 inhibitor U0126 did not alter the activation of β-catenin (B). Cytosolic extracts of osteoblasts treated with ESW in the presence of U0126 (with a final concentration of 20 μm) for 60 min prior to ESW were subjected to Western blotting. Phosphorylated β-catenin and β-catenin were probed with anti-phospho-β-catenin and β-catenin primary monoclonal antibodies, respectively. C, note that in comparison with the control, ESW exposure markedly elevated the activation of β-catenin. Further studies on the relationship between expression of integrins and β-catenin by transfection have shown that knocking out integrins led to base-line level expression of activation of β-catenin (B and D). D, summary of the results (mean ± S.E. (error bars), n = 4, triplicate in each experiment). a, p > 0.05 as compared with the control at the same period. b, p < 0.01 as compared with the control at the same period. c, p > 0.05 as compared with the ESW group. d, p < 0.01 as compared with the ESW group.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Activity Assay, Expressing, Phospho-proteomics, Activation Assay, Concentration Assay, Western Blot, Bioprocessing, Comparison, Control, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin ?1-mediated Expression of Phosphorylated Focal Adhesion Kinase

doi: 10.1074/jbc.M112.349811

Figure Lengend Snippet: Hypothetical model elucidating the regulation of phosphorylated FAK expression through an integrin α5 and β1-mediated MEK-ERK1/2-dependent pathway after ESWT. ESW directly stimulates integrin α5 and β1 mRNA expression inside the cell nucleus, and then the integrin protein expression is increased. Integrins induce MEK1/2 to phosphorylate ERK1/2, and then the activated ERK1/2 phosphorylates FAK and enhances its binding to the corresponding sites located in the adhesion sites, finally resulting in the enhancement of adhesion and migration. The Wnt/β-catenin signal pathway may also be involved in ESW-induced integrin-FAK signaling. The conformational activation of existing integrin α5/β1 complexes at the osteoblast surfaces may also be an additional potential mechanism that requires our further study.

Article Snippet: Membranes were probed with anti-integrin α5 and β1 antibodies (1:600 dilution; Santa Cruz Biotechnology, Inc.) at 4 °C overnight.

Techniques: Expressing, Binding Assay, Migration, Activation Assay