Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Baicalin Ameliorates Radiation-Induced Lung Injury by Inhibiting the CysLTs/CysLT1 Signaling Pathway

doi: 10.1155/2022/2765354

Figure Lengend Snippet: Immunofluorescence assay showed that BA reduced the expression of α -SMA in LTD4-induced type II AEC cells: (a) BA reduced the expression of α -SMA in LTD4-induced type II AEC cells (200-fold) and (b) BA reduced the fluorescence intensity of α -SMA in LTD4-induced type II AEC cells. LTD4: 150 nM LTD4-induced type II AEC cells and BA: baicalin. Compared with the control group, ▲ P < 0.05 and ▲▲ P < 0.01; compared with the LTD4 group, # P < 0.05 and ## P < 0.01. Data are expressed as the mean ± SD ( n = 3) and were analyzed by one-way ANOVA followed by Tukey analysis.

Article Snippet: Cells were incubated with primary antibody anti- α -SMA (cell signaling, 19245S) overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Fluorescence

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Baicalin Ameliorates Radiation-Induced Lung Injury by Inhibiting the CysLTs/CysLT1 Signaling Pathway

doi: 10.1155/2022/2765354

Figure Lengend Snippet: BA reduced the expression of CysLT1 mRNA, CysLT1, and α -SMA in lung tissue and type II AEC cells: (a) BA reduced the expression of CysLT1 mRNA in mice lung tissues and type II AEC cells and (b) and (c) BA reduced the expression of CysLT1 and α -SMA protein in mice lung tissues and type II AEC cells. IR: irradiation, BA: baicalin, DXM: dexamethasone, and LTD4: 150 nM LTD4-induced type II AEC cells. Compared with the control group, ▲ P < 0.05 and ▲▲ P < 0.01; compared with the IR or LTD4 group, # P < 0.05 and ## P < 0.01; and compared with the LTD4 + BA 80 μ g/mL group, ★ P < 0.05 and ★★ P < 0.01. Data are expressed as the mean ± SD ( n = 3) and were analyzed by one-way ANOVA followed by Tukey analysis.

Article Snippet: Cells were incubated with primary antibody anti- α -SMA (cell signaling, 19245S) overnight at 4°C.

Techniques: Expressing, Irradiation

Journal: Disease Markers

Article Title: Therapeutic Effects of 25-Hydroxyvitamin D on the Pathological Process of Benign Prostatic Hyperplasia: An In Vitro Evidence

doi: 10.1155/2021/4029470

Figure Lengend Snippet: 25-OH D ameliorates TGF- β 1 induces EMT of BPH-1 cells and proliferation of WPMY-1 cells via blocking TGF- β signaling. (a) Phosphorylation of Smad2 and Smad3 was detected by western blot assay after BPH-1 cells were treated with TGF- β 1 and 25-OH D. (b) Transcriptional activity of Smad2/Smad3/Smad4 complex was analyzed by luciferase assay after BPH-1 cells were treated with TGF- β 1 and 25-OH D. (c) Phosphorylation of Smad2 and Smad3 was detected by western blot assay after WPMY-1 cells were treated with TGF- β 1 and 25-OH D. (d) transcriptional activity of Smad2/Smad3/Smad4 complex was analyzed by luciferase assay after WPMY-1 cells were treated with TGF- β 1 and 25-OH D. (e) EMT of BPH-1 cells were evaluated by observing elongated fibroblast-like morphology with scattered distribution. (f) The mRNA and protein levels of EMT biomarkers including α -SMA, E-cadherin, and N-cadherin were detected by qRT-PCR and western blot assays. (g) Proliferation ability of WPMY-1 cells was assessed by CCK8 assays. (h) The mRNA and protein expressions of cell cycle-related genes including CCND1 and p21 were analyzed by qRT-PCR and western blot assays. ∗ P < 0.05.

Article Snippet: After washing, the membranes were then incubated with secondary antibodies at room temperature for 2 h. Antibodies used for the immunoreactivity were Smad2 [1 : 1000, #5339, Cell Signaling Technology (CST), USA]; Phospho-Smad2 (Ser465/Ser467) (1 : 1000, #18338, CST); Smad3 (1 : 1000, #9523, CST); Phospho-Smad3 (Ser423/425) (1 : 1000, #9520, CST); α -SMA (1 : 1000, #19245, CST); N-cadherin (1 : 1000, #13116, CST); E-cadherin (1 : 50, ab1416, Abcam, USA); CCND1 (1 : 200, ab16663, Abcam); p21 (1 : 1000, ab188224, Abcam); phospho-NF- κ B p65 (Ser536) (1 : 1000, #3033, CST); NF- κ B p65 (1 : 1000, #8242, CST); phospho-Stat3 (Tyr705) (1 : 2000, #9145, CST); Stat3 (1 : 1000, #9139, CST); Nfr2 (1 : 1000, #12721, CST); and GAPDH (1 : 2500, ab9485, Abcam).

Techniques: Blocking Assay, Western Blot, Activity Assay, Luciferase, Quantitative RT-PCR

Journal: Theranostics

Article Title: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis

doi: 10.7150/thno.72328

Figure Lengend Snippet: Mechanical ventilation induces pulmonary fibrosis. Mice lung tissue were acquired at Day 7 after 2 h of MV. A , lung injury was accessed and scored by Hematoxylin and Eosin staining. Original magnification × 200. Scale bars correspond to 100 µm, n = 12 per group. B , collagen deposition was assessed with Masson's trichrome staining and evaluated through Ashcroft fibrosis score. Original magnification × 200. Scale bars correspond to 100 µm, n = 12 per group. C-E , fibrosis was also quantified by determination of collagen-I α1 (COL1A1) ( C ), α smooth muscle actin (α-SMA) ( D ) in lung tissues (n = 3 in sham group and n = 9 in MV group) and type I procollagen carboxy-terminal peptide (PICP) levels in bronchoalveolar lavage fluid (BALF) ( E, n = 6 per group). F , lung tissues were stained with fluorophore-labeled antibodies against fibroblast marker fibronectin (FN) (Alexa Fluor 568, red) and collagen-I (fluorescein isothiocyanate, green). 4',6-diamidino-2- phenylindole (DAPI) stain was used to detect nuclei (blue). Original magnification × 400, Scale bars correspond to 20 µm. Data are expressed as means ± SEM. * p < 0.05, **p < 0.01, *** p < 0.001 (t test).

Article Snippet: The membrane was washed and blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 10% nonfat dry milk before overnight incubation with appropriate primary antibodies (integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), collagen-I α-1 (COL1A1, 72026S, CST, USA), α-smooth muscle actin (α-SMA) (CST, 19245), Lactate dehydrogenase (LDHA) (3582, CST, USA) and β-actin (8457, CST, USA)) in blocking buffer at 4 °C.

Techniques: Staining, Labeling, Marker

Journal: Theranostics

Article Title: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis

doi: 10.7150/thno.72328

Figure Lengend Snippet: PKM2 knockdown alleviates aerobic glycolysis and MV-induced pulmonary fibrosis. Mice were treated intratracheally with vector-AAV or shPKM-AAV 4 weeks before mechanical ventilation (MV), for the duration of 2 h. Lung tissues were harvested 7 days after intubation. A , lung fibrosis was quantified by Masson's trichrome staining. Original magnification × 200, Scale bars correspond to 100 µm, n = 6, 3, 6, 5, respectively in each group. B , pyruvate kinase M2 (PKM2), lactic dehydrogenase (LDHA), α smooth muscle actin (α-SMA) and collagen I α1 (COL1A1) expression was quantified by western blot analysis, n = 3 per group. C , lung tissues were stained with fluorophore labeled antibodies against fibroblast marker fibronectin (FN) (red) and collagen I (green), 4',6-diamidino-2- phenylindole (DAPI) stain was used to detect nuclei (blue), Original magnification × 400, Scale bars correspond to 20 µm. D and E , lactate (Lac) and type I procollagen carboxy-terminal peptide (PICP) in bronchoalveolar lavage fluid (BALF) was tested, n = 6, 3, 6, 5, respectively in each group. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01, *** p < 0.001 (ANOVA).

Article Snippet: The membrane was washed and blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 10% nonfat dry milk before overnight incubation with appropriate primary antibodies (integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), collagen-I α-1 (COL1A1, 72026S, CST, USA), α-smooth muscle actin (α-SMA) (CST, 19245), Lactate dehydrogenase (LDHA) (3582, CST, USA) and β-actin (8457, CST, USA)) in blocking buffer at 4 °C.

Techniques: Plasmid Preparation, Staining, Expressing, Western Blot, Labeling, Marker

Journal: Theranostics

Article Title: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis

doi: 10.7150/thno.72328

Figure Lengend Snippet: PKM2 inhibitor alleviates aerobic glycolysis and MV-induced pulmonary fibrosis. Mice were treated with PKM2 inhibitor shikonin 10 mg/kg in 100ul corn oil daily for 3 consecutive days before being subjected to MV for 2 h. Lung tissues were harvested 7 days after intubation. A , lung fibrosis was accessed by Masson's trichrome staining, Original magnification × 200, Scale bars correspond to 100 µm, n = 6, 3, 6, 3, respectively in each group. B , lactic dehydrogenase (LDHA), α smooth muscle actin (α-SMA) and collagen I α1 (COL1A1) expression was quantified by western blot analysis, n = 3 per group. C , lung tissues were stained with fluorophore-labeled antibodies against fibroblast marker fibronectin (FN) (red) and collagen I (green), 4',6-diamidino-2- phenylindole (DAPI) stain was used to detect nuclei (blue), original magnification × 400, Scale bars correspond to 20 µm. D and E , lactate (Lac) and type I procollagen carboxy-terminal peptide (PICP) levels in BALF was tested, n = 6, 3, 6, 3, respectively in each group. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01, *** p < 0.001 (ANOVA).

Article Snippet: The membrane was washed and blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 10% nonfat dry milk before overnight incubation with appropriate primary antibodies (integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), collagen-I α-1 (COL1A1, 72026S, CST, USA), α-smooth muscle actin (α-SMA) (CST, 19245), Lactate dehydrogenase (LDHA) (3582, CST, USA) and β-actin (8457, CST, USA)) in blocking buffer at 4 °C.

Techniques: Staining, Expressing, Western Blot, Labeling, Marker

Journal: Theranostics

Article Title: Integrin β3-PKM2 pathway-mediated aerobic glycolysis contributes to mechanical ventilation-induced pulmonary fibrosis

doi: 10.7150/thno.72328

Figure Lengend Snippet: Integrin β3 inhibition ameliorates PKM2-mediated aerobic glycolysis and MV-induced pulmonary fibrosis. A and B , both wild-type and integrin β3-deficient (β3 +/- ) mice were subjected to mechanical ventilation (MV) for 2 h. Lung tissues were harvested at day 7. A , lung fibrosis was quantified by Masson's trichrome staining, original magnification × 200, scale bars correspond to 100μm, n = 6, 5, 6, 6, respectively in each group. B , integrin β3, pyruvate kinase M2 (PKM2), lactic dehydrogenase (LDHA), collagen I α1 (COL1A1) and α smooth muscle actin (α-SMA) expression was quantified by western blot analysis, n = 3 per group. C and D , mice were treated intraperitoneally with integrin β3 inhibitor cilengitide 4 mg/kg once a day for 3 consecutive days before being subjected to MV for 2 h. Lung tissues were harvested 7 days after intubation. C , lung fibrosis was quantified by Masson's trichrome staining, original magnification × 200, scale bars correspond to 100 µm, n = 6 per group. D , integrin β3, PKM2, LDHA and COL1A1 expression was quantified by western blot analysis, n = 3 per group. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01, *** p < 0.001 (ANOVA).

Article Snippet: The membrane was washed and blocked with Tris-buffered saline (TBS) containing 0.1% Tween-20 and 10% nonfat dry milk before overnight incubation with appropriate primary antibodies (integrin β3 (SJ1909/NBP2-67416, Novus, USA), PKM2 (AF7244, R&D, USA), collagen-I α-1 (COL1A1, 72026S, CST, USA), α-smooth muscle actin (α-SMA) (CST, 19245), Lactate dehydrogenase (LDHA) (3582, CST, USA) and β-actin (8457, CST, USA)) in blocking buffer at 4 °C.

Techniques: Inhibition, Staining, Expressing, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Endothelial MicroRNA-483-3p Is Hypertension-Protective

doi: 10.1155/2022/3698219

Figure Lengend Snippet: EC-SMC cross talk via exosome-delivered miR-483-3p. (a) A representative electron microscopy image of HUVEC-derived exosomes. Scale bar, 100 nm. (b) A nanoparticle tracking analysis of the size distribution of exosomes. (c) Immunofluorescence images of HASMCs stained with α -SMA (red) and DAPI (blue). HASMCs were treated with HUVEC-derived exosomes (PKH67-label, green) for 0 or 24 hours. Laser scanning confocal images are shown. Scale bar, 20 μ m. (d) The qPCR analysis of miR-483-3p levels in HUVECs transfected with pre-miR-Ctrl or pre-miR-483-3p for 48 hours. (e) Exosomes isolated from HUVECs were identified by CD81 and CD63 immunostaining. (f) miR-483-3p levels in exosomes isolated from HUVECs transfected with pre-miR-Ctrl or pre-miR-483-3p. (g–i) HASMCs were incubated with HUVEC-derived exosomes for 24 hours. The qPCR analysis of (g) miR-483-3p and ACE1, TGF- β , and (h) CTGF in HASMCs. (i) Western blotting analysis of these proteins in HASMCs. Data are shown as mean ± SEM. Student's two-tailed t test was used for statistical analysis of data. ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: Cells were incubated with anti- α -SMA antibody (#19245, Cell Signaling Technology) overnight at 4°C, followed by a secondary antibody for 1 hour at room temperature.

Techniques: Electron Microscopy, Derivative Assay, Immunofluorescence, Staining, Transfection, Isolation, Immunostaining, Incubation, Western Blot, Two Tailed Test

Journal: BioMed Research International

Article Title: SIRT7 Regulates the Vascular Smooth Muscle Cells Proliferation and Migration via Wnt/ β -Catenin Signaling Pathway

doi: 10.1155/2018/4769596

Figure Lengend Snippet: The α -SMA and SIRT7 expression in ox-LDL-treated HAVSMCs. (a) Immunofluorescence staining was used to detect α -SMA in HAVSMCs stimulated with ox-LDL. Nuclei were stained with DAPI (blue), 200× magnification. (b) The protein expression level of α -SMA was measured by Western blot. ∗∗∗ P < 0.001 versus VSMCs. The protein level of SIRT7 was measured by Western blot (c) and RT-qPCR (d) in ox-LDL-treated HAVSMCs. ∗∗ P < 0.01 versus VSMCs. The expression level of SIRT7 was measured by Western blot (e) and RT-qPCR (f) after silencing of SIRT7 in ox-LDL-treated HAVSMCs. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 versus VSMCs; # P < 0.05 and ## P < 0.01 versus siRNA-NC. The expression level of SIRT7 was measured by Western blot (g) and RT-qPCR (h) after overexpression of SIRT7 in ox-LDL-treated HAVSMCs. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 versus VSMCs; ## P < 0.01 and ### P < 0.001 versus Vector-NC.

Article Snippet: Anti- α -SMA (1:1000; cat. no. 19245S), anti-SIRT7 (1:1000; cat. no. 5360S), anti- β -catenin (1:1000; cat. no. 8480T), anti-cyclin D1 (1:1000; cat. no. 3300T), and anti-GAPDH (1:1000; cat. no. 5174S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR, Over Expression, Plasmid Preparation

Journal: BioMed Research International

Article Title: SIRT7 Regulates the Vascular Smooth Muscle Cells Proliferation and Migration via Wnt/ β -Catenin Signaling Pathway

doi: 10.1155/2018/4769596

Figure Lengend Snippet: (a) The level of α -SMA after SIRT7 silencing or overexpression in ox-LDL-treated HAVSMCs was measured by immunofluorescence staining. Nuclei were stained with DAPI (blue), 200× magnification. (b) The protein expression level of α -SMA after SIRT7 silencing (b) or overexpression (c) in ox-LDL-treated HAVSMCs was assessed by Western blot. ∗ P < 0.05 and ∗∗ P < 0.01 versus VSMCs; # P < 0.05 versus siRNA-NC or Vector-NC.

Article Snippet: Anti- α -SMA (1:1000; cat. no. 19245S), anti-SIRT7 (1:1000; cat. no. 5360S), anti- β -catenin (1:1000; cat. no. 8480T), anti-cyclin D1 (1:1000; cat. no. 3300T), and anti-GAPDH (1:1000; cat. no. 5174S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Over Expression, Immunofluorescence, Staining, Expressing, Western Blot, Plasmid Preparation