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ATCC e coli
E Coli, supplied by ATCC, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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HRG-β1 induces phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: HRG-β1 induces phosphorylation of Smad2 in SK-BR-3 (a) and MCF7 (b) cells. (a) After 16 h of serum starvation in serum-free medium, SK-BR-3 cells were treated with 25 ng/ml of HRG-β1 for the indicated times. Immunoblots were probed with anti-phospho-Smad2 and anti-Smad2 antibodies. (b) The phosphorylation of Smad2 and total Smad2 were analyzed by western blotting in MCF7 cells. In all cases, β-actin was evaluated as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Article Snippet: For transfection, the cells were grown to confluence in 6-cm plates and a Smad2 siRNA (Santa Cruz Biotechnology Inc.) and a ErbB3 siRNA at 60 pmol (Santa Cruz Biotechnology Inc.) were transfected using a siRNA transfection reagent (Santa Cruz Biotechnology Inc.) according to the manufacturer’s instructions.

Techniques: Western Blot

Knockdown of ErbB3 suppresses HRG-β1-induced EMT in SK-BR-3 cells. The cells were transfected with control (Ctrl) or ErbB3 siRNAs and treated with 25 ng/ml of HRG-β1 for 24 h. The expressions of ErbB3, E-cadherin, fibronectin, phospho-Smad2, and Snail were analyzed by western blotting. β - actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: Knockdown of ErbB3 suppresses HRG-β1-induced EMT in SK-BR-3 cells. The cells were transfected with control (Ctrl) or ErbB3 siRNAs and treated with 25 ng/ml of HRG-β1 for 24 h. The expressions of ErbB3, E-cadherin, fibronectin, phospho-Smad2, and Snail were analyzed by western blotting. β - actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, significant difference.

Techniques: Transfection, Western Blot

HRG-β1 induces expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: HRG-β1 induces expression of Snail through phospho-Smad2 via PI3k/Akt in SK-BR-3 (a, b) and MCF7 (c, d) cells. (a, c) The cells were pretreated with vehicle (DMSO) as a control or 10 μM of phospho-Smad2 pharmacological inhibitors, PD169316 or SB203580 for 1 h, and then stimulated with HRG-β1 for 24 h. The inhibition of phospho-Smad2 by the inhibitors and total Smad2 were analyzed by western blotting in SK-BR-3 and MCF7 cells. (b) SK-BR-3 cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 prior to HRG-β1 stimulation for 24 h. The cells were harvested and immunoblots were analyzed with anti-phospho-Smad2, anti-Smad2, anti-Snail, and anti-β-actin antibodies. (d) Inhibition of HRG-β1-induced phospho-Smad2 and Snail expressions by 10 μM of LY294002 or SB203580 without affecting total Smad2 and constitutive β-actin expressions in MCF7 cells. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Techniques: Expressing, Inhibition, Western Blot

HRG-β1 induces nuclear colocalization of phospho-Smad2 and Snail. Immunofluorescence analyses were performed for the nuclear colocalization of phospho-Smad2 and Snail in SK-BR-3 cells. Cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 for 1 h prior to stimulation with 25 ng/ml of HRG-β1. After incubation for a further 24 h, phospho-Smad2 (red) and Snail (green) were observed under a confocal laser scanning microscope and the nuclear DNA was stained with DAPI (blue; magnification, ×200). The lower graphs show the fluorescence intensities of phospho-Smad2 and Snail as percentages compared with control cells in three independent experiments involving SK-BR-3 (left) and MCF7 (right) cells. * P < 0.05, ** P < 0.01, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: HRG-β1 induces nuclear colocalization of phospho-Smad2 and Snail. Immunofluorescence analyses were performed for the nuclear colocalization of phospho-Smad2 and Snail in SK-BR-3 cells. Cells were pretreated with vehicle or 10 μM of LY294002 or PD169316 for 1 h prior to stimulation with 25 ng/ml of HRG-β1. After incubation for a further 24 h, phospho-Smad2 (red) and Snail (green) were observed under a confocal laser scanning microscope and the nuclear DNA was stained with DAPI (blue; magnification, ×200). The lower graphs show the fluorescence intensities of phospho-Smad2 and Snail as percentages compared with control cells in three independent experiments involving SK-BR-3 (left) and MCF7 (right) cells. * P < 0.05, ** P < 0.01, significant difference.

Techniques: Immunofluorescence, Incubation, Laser-Scanning Microscopy, Staining, Fluorescence

HRG-β1 induces EMT through phospho-Smad2-mediated Snail via PI3k/Akt signaling in SK-BR-3 (a) and MCF7 (b) cells. (a) The cells were pretreated for 1 h with vehicle, 10 μM of LY294002, or PD169316 alone or a combination of LY294002 and PD169316 and then treated with 25 ng/ml of HRG-β1. The expressions of vimentin and fibronectin were identified by western blotting. ( b ) The cells were treated with 25 ng/ml of HRG-β1 after pretreatment with 10 μM of LY294002 or SB203580 alone or a combination of LY294002 and SB203580. Immunoblots were probed with anti-vimentin and anti-fibronectin antibodies. In all cases, β-actin served as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: HRG-β1 induces EMT through phospho-Smad2-mediated Snail via PI3k/Akt signaling in SK-BR-3 (a) and MCF7 (b) cells. (a) The cells were pretreated for 1 h with vehicle, 10 μM of LY294002, or PD169316 alone or a combination of LY294002 and PD169316 and then treated with 25 ng/ml of HRG-β1. The expressions of vimentin and fibronectin were identified by western blotting. ( b ) The cells were treated with 25 ng/ml of HRG-β1 after pretreatment with 10 μM of LY294002 or SB203580 alone or a combination of LY294002 and SB203580. Immunoblots were probed with anti-vimentin and anti-fibronectin antibodies. In all cases, β-actin served as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, significant difference.

Techniques: Western Blot

Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β - actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, significant difference.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: Knockdown of Smad2 suppresses HRG-β1-induced expressions of Snail and fibronectin in SK-BR-3 (a) and MCF7 (b) cells. (a, b) The cells were transfected with control or Smad2 siRNAs prior to treatment with 25 ng/ml of HRG-β1. After incubation for a further 24 h, the expressions of phospho-Smad2, Smad2, Snail, and fibronectin were analyzed by western blotting. β - actin was reprobed as a loading control. Data represent the means ± SD of three independent experiments. * P < 0.05, ** P < 0.01, significant difference.

Techniques: Transfection, Incubation, Western Blot

HRG-β1-induces cancer cell migration and invasion through Smad2 activation in SK-BR-3 (a, c) and MCF7 (b, d) cells. (a, b) The motility of each cell type was assessed by wound healing assays. A scratch was made across confluent monolayers using a plastic tip and the cells were then pretreated with 10 μM of LY294002 and PD169316 or SB203580 prior to stimulation with HRG-β1. After 24 h of incubation, the migrated cells were monitored using a light microscope. Data were analyzed as percentages of the control cells in three independent experiments. * P < 0.05, significant difference. (c, d) A matrigel invasion assay was used to quantify cell invasion. After 24 h of transfection, the cells were seeded into upper chambers and incubated for 48 h in the presence of 25 ng/ml of HRG-β1. Then, the cells that invaded into the lower surface were photographed under a light microscope, with × 200 magnification. Data were analyzed as the percentage of the control of the three independent experiments. * P < 0.05 and ** P < 0.01 were considered significant.

Journal: BMC Cancer

Article Title: HRG-β1-driven ErbB3 signaling induces epithelial–mesenchymal transition in breast cancer cells

doi: 10.1186/1471-2407-13-383

Figure Lengend Snippet: HRG-β1-induces cancer cell migration and invasion through Smad2 activation in SK-BR-3 (a, c) and MCF7 (b, d) cells. (a, b) The motility of each cell type was assessed by wound healing assays. A scratch was made across confluent monolayers using a plastic tip and the cells were then pretreated with 10 μM of LY294002 and PD169316 or SB203580 prior to stimulation with HRG-β1. After 24 h of incubation, the migrated cells were monitored using a light microscope. Data were analyzed as percentages of the control cells in three independent experiments. * P < 0.05, significant difference. (c, d) A matrigel invasion assay was used to quantify cell invasion. After 24 h of transfection, the cells were seeded into upper chambers and incubated for 48 h in the presence of 25 ng/ml of HRG-β1. Then, the cells that invaded into the lower surface were photographed under a light microscope, with × 200 magnification. Data were analyzed as the percentage of the control of the three independent experiments. * P < 0.05 and ** P < 0.01 were considered significant.

Techniques: Migration, Activation Assay, Incubation, Light Microscopy, Invasion Assay, Transfection

GC-derived TGF-β1 induces Smad3-dependent PD-1 expression and Smad2-dependent CD8+ T cell dysfunction. (A) CD8+ T cells were purified from PBMCs, pretreated with SIS3 or DMSO for 1 hour, and then exposed to 30% TSN for 72 hours in the presence of anti-CD3 and anti-CD28 antibodies. The expression of PD-1, IFN-γ, TNF-α, granzyme B and perforin of these CD8+ T cells were analyzed. (B) Statistical analysis of PD-1+ cell percentages and mean fluorescence intensity of CD8+ T cells from different groups (n=5). (C) Statistical analysis of IFN-γ+, TNF-α+, granzyme B+ and perforin+ cell percentages in CD8+ T cells from different groups (n=5). (D) CD8+ T cells transduced with lentiviral particles containing shSmad2 or shNC were exposed to 30% TSN for 72 hours in the presence of anti-CD3 and anti-CD28 antibodies, and then the expression of IFN-γ, TNF-α, granzyme B and perforin in these CD8+ T cells were analyzed by flow cytometry. (E) Statistical analysis of IFN-γ+, TNF-α+, granzyme B+ and perforin+ cell percentages in CD8+ T cells from different groups (n=4). *p<0.05, **p<0.01: Student’s t test. GC, gastric cancer; PD-1, programmed cell death protein 1; TSN, culture supernatant from digested primary GC tumor tissues.

Journal: Journal for Immunotherapy of Cancer

Article Title: PD-1 does not mark tumor-infiltrating CD8+ T cell dysfunction in human gastric cancer

doi: 10.1136/jitc-2019-000422

Figure Lengend Snippet: GC-derived TGF-β1 induces Smad3-dependent PD-1 expression and Smad2-dependent CD8+ T cell dysfunction. (A) CD8+ T cells were purified from PBMCs, pretreated with SIS3 or DMSO for 1 hour, and then exposed to 30% TSN for 72 hours in the presence of anti-CD3 and anti-CD28 antibodies. The expression of PD-1, IFN-γ, TNF-α, granzyme B and perforin of these CD8+ T cells were analyzed. (B) Statistical analysis of PD-1+ cell percentages and mean fluorescence intensity of CD8+ T cells from different groups (n=5). (C) Statistical analysis of IFN-γ+, TNF-α+, granzyme B+ and perforin+ cell percentages in CD8+ T cells from different groups (n=5). (D) CD8+ T cells transduced with lentiviral particles containing shSmad2 or shNC were exposed to 30% TSN for 72 hours in the presence of anti-CD3 and anti-CD28 antibodies, and then the expression of IFN-γ, TNF-α, granzyme B and perforin in these CD8+ T cells were analyzed by flow cytometry. (E) Statistical analysis of IFN-γ+, TNF-α+, granzyme B+ and perforin+ cell percentages in CD8+ T cells from different groups (n=4). *p<0.05, **p<0.01: Student’s t test. GC, gastric cancer; PD-1, programmed cell death protein 1; TSN, culture supernatant from digested primary GC tumor tissues.

Article Snippet: In some cases, CD8+ T cells were pretreated with 10 µM of the Smad3-specific inhibitor SIS3 (MedChem Express, Monmouth Junction, NJ) or dimethyl sulfoxide (DMSO) for 1 hour following stimulation with 30% TSN, or CD8+ T cells were activated with precoated anti-CD3 (2 µg/mL) and anti-CD28 (1 µg/mL) antibodies for 24 hours, and subsequently lentiviral particles containing Smad2 shRNA (shSmad2, Santa Cruz Biotechnology) or control shRNA (shNC, Santa Cruz Biotechnology) were added.

Techniques: Derivative Assay, Expressing, Purification, Fluorescence, Transduction, Flow Cytometry

Schematic representation of experimental animal models obtained for a period of 4 months. Golden Syrian hamsters (67 males and 3 months old) were divided into seven experimental groups: (1) control (C group) ; (2) simultaneously hypertensive–hyperlipidemic (HH group) ; (3,4) HH hamsters with retro-orbital sinus injection containing 100 μg/ml EVs from both ADSCs and BM-MSCs ( HH-EVs (ADSCs) group and HH-EVs (MSCs) group )) ; (5,6) HH hamsters with retro-orbital sinus injection containing 100 μg/ml EVs (from ADSCs or BM-MSCs) transfected with 100 nM Smad2/3 siRNA (HH-EVs (ADSCs)+Smad2/3 siRNA group and HH-EVs (MSCs)+ Smad2/3 siRNA group) ; (7) HH hamsters with subcutaneous injection containing 100 nM Smad2/3 siRNA (HH-Smad2/3 siRNA group) . The HH group was obtained by combining the atherogenic and high-salt diet.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Schematic representation of experimental animal models obtained for a period of 4 months. Golden Syrian hamsters (67 males and 3 months old) were divided into seven experimental groups: (1) control (C group) ; (2) simultaneously hypertensive–hyperlipidemic (HH group) ; (3,4) HH hamsters with retro-orbital sinus injection containing 100 μg/ml EVs from both ADSCs and BM-MSCs ( HH-EVs (ADSCs) group and HH-EVs (MSCs) group )) ; (5,6) HH hamsters with retro-orbital sinus injection containing 100 μg/ml EVs (from ADSCs or BM-MSCs) transfected with 100 nM Smad2/3 siRNA (HH-EVs (ADSCs)+Smad2/3 siRNA group and HH-EVs (MSCs)+ Smad2/3 siRNA group) ; (7) HH hamsters with subcutaneous injection containing 100 nM Smad2/3 siRNA (HH-Smad2/3 siRNA group) . The HH group was obtained by combining the atherogenic and high-salt diet.

Article Snippet: Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific) was used to transfect EVs with a siRNA-targeting Smad2/3 (100 nM final concentration) (Santa Cruz Biotechnology, Inc.) according to the manufacturer’s protocol.

Techniques: Injection, Transfection

Clinical characteristics and biochemical parameters of the seven experimental animal groups, control (C); simultaneously hypertensive–hyperlipidemic (HH); HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3 siRNA, after 16 weeks of standard diet or atherogenic diet, and at 14 weeks post-treatment with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA during the diet-induced atherosclerotic process. Data are means ± SD of duplicate determinations. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01, * p < 0.05 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Two-way ANOVA and Bonferroni post-test were applied.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Clinical characteristics and biochemical parameters of the seven experimental animal groups, control (C); simultaneously hypertensive–hyperlipidemic (HH); HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3 siRNA, after 16 weeks of standard diet or atherogenic diet, and at 14 weeks post-treatment with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA during the diet-induced atherosclerotic process. Data are means ± SD of duplicate determinations. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01, * p < 0.05 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Two-way ANOVA and Bonferroni post-test were applied.

Techniques: Transfection

Changes in the blood flow and structure of the thoracic aorta and carotid artery were isolated from the investigated experimental groups (C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, and HH-EVs(MSCs)+Smad2/3siRNA) as a measure of vascular rigidity. (A) Representative B-mode recordings, which highlight the wall thickness and the inner diameter of the thoracic aorta; (B) graphical representation of wall thickness (mm) (B.1) and inner diameter (mm) (B.2) in the case of the thoracic aorta; (C) representative records obtained in M-mode, which highlight the diameter in systole and diastole of the thoracic aorta; (D) graphical representation of thoracic aortic distensibility (mm); (E) representative recordings obtained in pulsed Doppler-mode, which highlight the velocity time integral (VTI) and velocity (Vel) of the thoracic aorta; (F) graphical representation of velocity (mm/s) (F.1) and velocity time integral (mm) (F.2) at the level of the thoracic aorta; (G) representative B-mode recordings, which highlight the wall thickness and the inner diameter of the carotid artery; (H) graphical representation of wall thickness (mm) (H.1) and inner diameter (mm) (H.2) in the case of the carotid artery. Data are shown as the mean ± SD of each experimental group after 4 months of diet and treatment. The statistical significance, noticeably different, is represented as *** p < 0.005, ** p < 0.01, * p < 0.05 versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Changes in the blood flow and structure of the thoracic aorta and carotid artery were isolated from the investigated experimental groups (C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, and HH-EVs(MSCs)+Smad2/3siRNA) as a measure of vascular rigidity. (A) Representative B-mode recordings, which highlight the wall thickness and the inner diameter of the thoracic aorta; (B) graphical representation of wall thickness (mm) (B.1) and inner diameter (mm) (B.2) in the case of the thoracic aorta; (C) representative records obtained in M-mode, which highlight the diameter in systole and diastole of the thoracic aorta; (D) graphical representation of thoracic aortic distensibility (mm); (E) representative recordings obtained in pulsed Doppler-mode, which highlight the velocity time integral (VTI) and velocity (Vel) of the thoracic aorta; (F) graphical representation of velocity (mm/s) (F.1) and velocity time integral (mm) (F.2) at the level of the thoracic aorta; (G) representative B-mode recordings, which highlight the wall thickness and the inner diameter of the carotid artery; (H) graphical representation of wall thickness (mm) (H.1) and inner diameter (mm) (H.2) in the case of the carotid artery. Data are shown as the mean ± SD of each experimental group after 4 months of diet and treatment. The statistical significance, noticeably different, is represented as *** p < 0.005, ** p < 0.01, * p < 0.05 versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test.

Techniques: Isolation

Representative images with myograph recordings at selected time points: for the contraction function to NA (10 −8 M ÷ 10 −4 M) and relaxation to ACh (10 −8 M ÷ 10 −4 M) in the thoracic aorta (red) and carotid artery (purple) in all investigated experimental groups: C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA. Images were recorded with LabChart 7 software.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Representative images with myograph recordings at selected time points: for the contraction function to NA (10 −8 M ÷ 10 −4 M) and relaxation to ACh (10 −8 M ÷ 10 −4 M) in the thoracic aorta (red) and carotid artery (purple) in all investigated experimental groups: C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA. Images were recorded with LabChart 7 software.

Techniques: Software

Measures of vascular reactivity of the thoracic aorta (left) and carotid artery (right) explanted from all hamster groups (C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA) by using the myograph technique, in terms of (A) contraction to NA and (B) relaxation to ACh. Maximal contractile force developed by the thoracic aorta and carotid artery was measured to be 10 −4 M NA, and maximal relaxation values were recorded to be 10 −5 M ACh for the thoracic aorta and 10 −6 M ACh for the carotid artery. Data are mean ± SD of four independent experiments for each investigated treated group and five independent experiments for control and HH groups. The statistical significance, noticeably different, was represented as *** p < 0.005 and * p < 0.05 versus control group and ### p < 0.005 and ## p < 0.01 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test. Enhanced plasma TGF-β1 and AngII levels in atherosclerosis are reduced after the administration of EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3 siRNA.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Measures of vascular reactivity of the thoracic aorta (left) and carotid artery (right) explanted from all hamster groups (C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA) by using the myograph technique, in terms of (A) contraction to NA and (B) relaxation to ACh. Maximal contractile force developed by the thoracic aorta and carotid artery was measured to be 10 −4 M NA, and maximal relaxation values were recorded to be 10 −5 M ACh for the thoracic aorta and 10 −6 M ACh for the carotid artery. Data are mean ± SD of four independent experiments for each investigated treated group and five independent experiments for control and HH groups. The statistical significance, noticeably different, was represented as *** p < 0.005 and * p < 0.05 versus control group and ### p < 0.005 and ## p < 0.01 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test. Enhanced plasma TGF-β1 and AngII levels in atherosclerosis are reduced after the administration of EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3 siRNA.

Techniques: Transfection

Analysis of plasma TGF-β1 and AngII levels by the enzyme-linked immunosorbent assay (ELISA) method, for all experimental groups: C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA. The measurements were performed in triplicate, and the results were depicted as mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01 versus control group and ### p < 0.005, # p < 0.05 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Analysis of plasma TGF-β1 and AngII levels by the enzyme-linked immunosorbent assay (ELISA) method, for all experimental groups: C, HH, HH-EVs(ADSCs), HH-EVs(MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA. The measurements were performed in triplicate, and the results were depicted as mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01 versus control group and ### p < 0.005, # p < 0.05 versus HH group. The values were calculated by two-way ANOVA and Bonferroni post-test.

Techniques: Enzyme-linked Immunosorbent Assay

Representative immunofluorescence images for the evaluation of inflammatory markers specific to vascular dysfunction after 4 months of the hyperlipemic–hypertensive diet and the stem cell-derived EV-based treatment or siRNA-based treatment. The thin cryosections from the thoracic aorta (on the left) and carotid artery (on the right) harvested from all experimental groups (C, HH, HH-EVs (ADSCs), HH-EVs (MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA) were immuno-labeled for the following: 1) structural proteins: collagen type I (COL1A1) alpha smooth muscle actin (α-SMA), and connexin 43 (Cx43); 2) proteins involved in cell adhesion and vascular remodeling: matrix metalloproteinase-2 (MMP-2) and vascular cell adhesion molecule-1 (VCAM-1); 3) immune cell infiltrate: T cells (CD3e+), total macrophages (CD68 + ), and M1 macrophages (MHC-II+); and 4) cytosolic ROS production (dihydroethidium (DHE) was oxidized by cytosolic ROS to fluorescent ethidium bromide that intercalates DNA yielding a bright red nuclear fluorescence). Nuclei were shown in blue fluorescence by DAPI dye staining. Each experiment point was performed in triplicate, from two different sets of experiments. A total of five different microscopic fields for each experimental point were analyzed. Total magnification: ×20. The images were quantified using the ImageJ program.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Representative immunofluorescence images for the evaluation of inflammatory markers specific to vascular dysfunction after 4 months of the hyperlipemic–hypertensive diet and the stem cell-derived EV-based treatment or siRNA-based treatment. The thin cryosections from the thoracic aorta (on the left) and carotid artery (on the right) harvested from all experimental groups (C, HH, HH-EVs (ADSCs), HH-EVs (MSCs), HH-EVs(ADSCs)+Smad2/3siRNA, HH-EVs(MSCs)+Smad2/3siRNA, and HH-Smad2/3siRNA) were immuno-labeled for the following: 1) structural proteins: collagen type I (COL1A1) alpha smooth muscle actin (α-SMA), and connexin 43 (Cx43); 2) proteins involved in cell adhesion and vascular remodeling: matrix metalloproteinase-2 (MMP-2) and vascular cell adhesion molecule-1 (VCAM-1); 3) immune cell infiltrate: T cells (CD3e+), total macrophages (CD68 + ), and M1 macrophages (MHC-II+); and 4) cytosolic ROS production (dihydroethidium (DHE) was oxidized by cytosolic ROS to fluorescent ethidium bromide that intercalates DNA yielding a bright red nuclear fluorescence). Nuclei were shown in blue fluorescence by DAPI dye staining. Each experiment point was performed in triplicate, from two different sets of experiments. A total of five different microscopic fields for each experimental point were analyzed. Total magnification: ×20. The images were quantified using the ImageJ program.

Techniques: Immunofluorescence, Derivative Assay, Labeling, Fluorescence, Staining

Quantification of the stained areas of inflammatory markers from the fluorescence images of the thoracic aorta and carotid artery sections collected from all investigated animal groups. Results were expressed as mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01, * p < 0.05 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Two-way ANOVA and Bonferroni post-test were applied.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Quantification of the stained areas of inflammatory markers from the fluorescence images of the thoracic aorta and carotid artery sections collected from all investigated animal groups. Results were expressed as mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01, * p < 0.05 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Two-way ANOVA and Bonferroni post-test were applied.

Techniques: Staining, Fluorescence

Representative Western blotting images of the expression levels of pATF-2, ATF-2, pSMAD2/3, SMAD2/3, NF-kBp50, NF-kBp65, and β-actin in both thoracic aorta (left) and carotid artery (right) explanted from all seven experimental animal groups.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Representative Western blotting images of the expression levels of pATF-2, ATF-2, pSMAD2/3, SMAD2/3, NF-kBp50, NF-kBp65, and β-actin in both thoracic aorta (left) and carotid artery (right) explanted from all seven experimental animal groups.

Techniques: Western Blot, Expressing

Western Blot analysis for relative expression of specific pro-inflammatory molecules (proteins): pATF-2, ATF-2, pSMAD2/3, SMAD2/3, and NF-kBp50/p65. Histograms show a quantitative representation of the protein levels obtained from all investigated groups of four independent experiments after 4 months of diet and treatment. Each value represents the mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Statistical analysis was conducted using two-way ANOVA and Bonferroni post-test. The gray intensity of related proteins was analyzed by the TotalLab TL120 program. The housekeeping β-actin protein was used as an internal control for protein normalization and monitor for equal loading. Note that the β-actin expression fluctuated upon the treatment or under physiological and pathological conditions.

Journal: Frontiers in Cell and Developmental Biology

Article Title: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players

doi: 10.3389/fcell.2022.817180

Figure Lengend Snippet: Western Blot analysis for relative expression of specific pro-inflammatory molecules (proteins): pATF-2, ATF-2, pSMAD2/3, SMAD2/3, and NF-kBp50/p65. Histograms show a quantitative representation of the protein levels obtained from all investigated groups of four independent experiments after 4 months of diet and treatment. Each value represents the mean ± SD. The statistical significance, noticeably different, was represented as *** p < 0.005, ** p < 0.01 values versus control group and ### p < 0.005, ## p < 0.01, # p < 0.05 values versus HH group. Statistical analysis was conducted using two-way ANOVA and Bonferroni post-test. The gray intensity of related proteins was analyzed by the TotalLab TL120 program. The housekeeping β-actin protein was used as an internal control for protein normalization and monitor for equal loading. Note that the β-actin expression fluctuated upon the treatment or under physiological and pathological conditions.

Techniques: Western Blot, Expressing

Journal: Cancer Science

Article Title: Circ_0002623 promotes bladder cancer progression by regulating the miR‐1276/SMAD2 axis

doi: 10.1111/cas.15274

Figure Lengend Snippet: Primer sequences

Article Snippet: The siRNAs targeting SMAD2 were bought from Santa Cruz Biotechnology.

Techniques: Sequencing

SMAD2 is a target of miR‐1276. A, Three databases, StarBase, TargetScan, and miRDB, were used to comprehensively analyze the potential target genes of miR‐1276. B, KEGG analysis of the signaling pathways where miR‐1276 target genes are enriched. C, The binding site between miR‐1276 and SMAD2 was predicted by bioinformatics analysis. D, Dual‐luciferase reporter gene assay validated the binding site between miR‐1276 and SMAD2. E and F, Quantitative RT‐PCR and Western blot were used to detect the regulatory effect of miR‐1276 on SMAD2 expression. G, Quantitative RT‐PCR was used to detect SMAD2 expression in 32 pairs of bladder cancer (BCa) tissues and the paracancerous tissues. H, The GEPIA2 database was used to analyze the relationship between SMAD2 expression and BCa patients’ overall survival. *** p < .001

Journal: Cancer Science

Article Title: Circ_0002623 promotes bladder cancer progression by regulating the miR‐1276/SMAD2 axis

doi: 10.1111/cas.15274

Figure Lengend Snippet: SMAD2 is a target of miR‐1276. A, Three databases, StarBase, TargetScan, and miRDB, were used to comprehensively analyze the potential target genes of miR‐1276. B, KEGG analysis of the signaling pathways where miR‐1276 target genes are enriched. C, The binding site between miR‐1276 and SMAD2 was predicted by bioinformatics analysis. D, Dual‐luciferase reporter gene assay validated the binding site between miR‐1276 and SMAD2. E and F, Quantitative RT‐PCR and Western blot were used to detect the regulatory effect of miR‐1276 on SMAD2 expression. G, Quantitative RT‐PCR was used to detect SMAD2 expression in 32 pairs of bladder cancer (BCa) tissues and the paracancerous tissues. H, The GEPIA2 database was used to analyze the relationship between SMAD2 expression and BCa patients’ overall survival. *** p < .001

Article Snippet: The siRNAs targeting SMAD2 were bought from Santa Cruz Biotechnology.

Techniques: Binding Assay, Luciferase, Reporter Gene Assay, Quantitative RT-PCR, Western Blot, Expressing

The pathological evaluation of the metastatic nodules in the lung tissues of the nude mice

Journal: Cancer Science

Article Title: Circ_0002623 promotes bladder cancer progression by regulating the miR‐1276/SMAD2 axis

doi: 10.1111/cas.15274

Figure Lengend Snippet: The pathological evaluation of the metastatic nodules in the lung tissues of the nude mice

Article Snippet: The siRNAs targeting SMAD2 were bought from Santa Cruz Biotechnology.

Techniques:

Circ_0002623 promotes the activation of the TGF‐β and Wnt signaling pathways by regulating SMAD2. A, Gene set enrichment analysis (GSEA) was performed to predict the biological effects of SMAD2 in bladder cancer (BCa) tissues, and it showed that SMAD2 high expression was associated with the activation of the TGF‐β and Wnt signaling pathways. B, Western blotting was used to detect the regulatory effects of circ_0002623, miR‐1276, and SMAD2 on the expression levels of TGF‐β and WNT1

Journal: Cancer Science

Article Title: Circ_0002623 promotes bladder cancer progression by regulating the miR‐1276/SMAD2 axis

doi: 10.1111/cas.15274

Figure Lengend Snippet: Circ_0002623 promotes the activation of the TGF‐β and Wnt signaling pathways by regulating SMAD2. A, Gene set enrichment analysis (GSEA) was performed to predict the biological effects of SMAD2 in bladder cancer (BCa) tissues, and it showed that SMAD2 high expression was associated with the activation of the TGF‐β and Wnt signaling pathways. B, Western blotting was used to detect the regulatory effects of circ_0002623, miR‐1276, and SMAD2 on the expression levels of TGF‐β and WNT1

Article Snippet: The siRNAs targeting SMAD2 were bought from Santa Cruz Biotechnology.

Techniques: Activation Assay, Expressing, Western Blot

Graphic abstract: circ_0002623 increases SMAD2 expression via sponging miR‐1276, thereby promoting the malignant phenotypes of bladder cancer (BCa) cells

Journal: Cancer Science

Article Title: Circ_0002623 promotes bladder cancer progression by regulating the miR‐1276/SMAD2 axis

doi: 10.1111/cas.15274

Figure Lengend Snippet: Graphic abstract: circ_0002623 increases SMAD2 expression via sponging miR‐1276, thereby promoting the malignant phenotypes of bladder cancer (BCa) cells

Article Snippet: The siRNAs targeting SMAD2 were bought from Santa Cruz Biotechnology.

Techniques: Expressing

(A, B) ST2 cells were pretreated with indicated concentrations of A83-01 for 1 h. Cells were then incubated with 1α,25(OH) 2 D 3 ( D 3 ) (10 −7 M), dexamethasone (Dex) (10 −7 M), and TGF-β1 (2.0 ng/mL) for 12 h (A) and 24 h (B). The mRNA (A) and the protein (B) level of RANKL were detected using real-time RT-PCR and Western blot analysis, respectively. (B) Bars represent means with the standard deviation of relative band intensities normalized to changes in the β‐actin from independent triplicate samples. (C) ST2 cells were transfected with either control siRNA or smad2/3 siRNA and then cultured for 24 h. The mRNA levels of smad2 and smad3 were assessed using real-time RT-PCR. (D) Transfected ST2 cells were incubated for 24 h, and stimulated with D 3 (10 −7 M), Dex (10 −7 M), and TGF-β1 (2.0 ng/mL) for 12 h. The mRNA level of rankl was assessed using real-time RT-PCR. Data are displayed as mean ± S.D. of three independent cultures. n . s . = not significant, *P < 0.05, **P < 0.01, ***P < 0.0001.

Journal: PLoS ONE

Article Title: Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-β1 via the ubiquitin-proteasome system

doi: 10.1371/journal.pone.0262612

Figure Lengend Snippet: (A, B) ST2 cells were pretreated with indicated concentrations of A83-01 for 1 h. Cells were then incubated with 1α,25(OH) 2 D 3 ( D 3 ) (10 −7 M), dexamethasone (Dex) (10 −7 M), and TGF-β1 (2.0 ng/mL) for 12 h (A) and 24 h (B). The mRNA (A) and the protein (B) level of RANKL were detected using real-time RT-PCR and Western blot analysis, respectively. (B) Bars represent means with the standard deviation of relative band intensities normalized to changes in the β‐actin from independent triplicate samples. (C) ST2 cells were transfected with either control siRNA or smad2/3 siRNA and then cultured for 24 h. The mRNA levels of smad2 and smad3 were assessed using real-time RT-PCR. (D) Transfected ST2 cells were incubated for 24 h, and stimulated with D 3 (10 −7 M), Dex (10 −7 M), and TGF-β1 (2.0 ng/mL) for 12 h. The mRNA level of rankl was assessed using real-time RT-PCR. Data are displayed as mean ± S.D. of three independent cultures. n . s . = not significant, *P < 0.05, **P < 0.01, ***P < 0.0001.

Article Snippet: Specific siRNA (Santa Cruz Biotechnology) was used to knockdown smad2 and smad3 expression in ST2 cells.

Techniques: Incubation, Quantitative RT-PCR, Western Blot, Standard Deviation, Transfection, Cell Culture

(A, B) ST2 cells were treated with TGF-β1 (2.0 ng/mL) for indicated time periods. (A) RXR-α protein level was detected via Western blot analysis. β-actin served as the loading control. (B) The mRNA level of rxr-α was assessed via real-time RT-PCR. (C, D) ST2 cells were pretreated with A83-01 (2.0 μM) for 1 h (C) or transfected with either control siRNA or smad2/3 siRNA (D), prior to stimulation with TGF-β1 (2.0 ng/mL) for 12 h. RXR-α protein level was detected by Western blot analysis. β-actin served as the loading control. (A, C, D) Bars represent means with the standard deviation of relative band intensities normalized to changes in the β‐actin from independent triplicate samples. Data are displayed as mean ± S.D. of three independent cultures. n . s . = not significant, *P < 0.05, ***P < 0.0001.

Journal: PLoS ONE

Article Title: Mechanisms involved in suppression of osteoclast supportive activity by transforming growth factor-β1 via the ubiquitin-proteasome system

doi: 10.1371/journal.pone.0262612

Figure Lengend Snippet: (A, B) ST2 cells were treated with TGF-β1 (2.0 ng/mL) for indicated time periods. (A) RXR-α protein level was detected via Western blot analysis. β-actin served as the loading control. (B) The mRNA level of rxr-α was assessed via real-time RT-PCR. (C, D) ST2 cells were pretreated with A83-01 (2.0 μM) for 1 h (C) or transfected with either control siRNA or smad2/3 siRNA (D), prior to stimulation with TGF-β1 (2.0 ng/mL) for 12 h. RXR-α protein level was detected by Western blot analysis. β-actin served as the loading control. (A, C, D) Bars represent means with the standard deviation of relative band intensities normalized to changes in the β‐actin from independent triplicate samples. Data are displayed as mean ± S.D. of three independent cultures. n . s . = not significant, *P < 0.05, ***P < 0.0001.

Article Snippet: Specific siRNA (Santa Cruz Biotechnology) was used to knockdown smad2 and smad3 expression in ST2 cells.

Techniques: Western Blot, Quantitative RT-PCR, Transfection, Standard Deviation

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