Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of the TGF-β/BMP Signaling Pathway on the Proliferation of Yak Pulmonary Artery Smooth Muscle Cells under Hypoxic Conditions

doi: 10.3390/ani14142072

Figure Lengend Snippet: Impact of hypoxia on BMP/TGF-β signaling pathway-related protein expression and proliferation in yak PASMCs. ( A – C ) Western blot analysis of HIF-1α and HIF-2α protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( D – G ) Western blot analysis of TGF-β/Smad2/3 signaling pathway-related protein expression under hypoxia conditions, with a densitometric analysis performed using ImageJ. ( H – J ) Western blot analysis of BMP/Smad1/5 signaling pathway-related protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( K – M ) Western blot analysis of BCL-2 and PCNA protein expression levels under hypoxic conditions, with a densitometric analysis performed using ImageJ. ** p < 0.01, * 0.01 < p < 0.05, ns indicates insignificant difference.

Article Snippet: The membrane was blocked at room temperature with 50 g·L −1 non-fat dry milk powder for 3 h. Then, the samples were incubated overnight at 4 °C with primary antibodies for HIF-1α (1:1000; ab16066, Abcam, Cambridge, MA, USA), HIF-2α (1:800; AF6362, Affinity, Jiangsu, China), TGF-β1 (1:500; bs-0086R, Bioss, Beijing, China), Smad2 (1:500; bs-0718R, Bioss, Beijing, China), Smad3 (1:500; bs-8853R, Bioss, Beijing, China), phosphorylated Smad2 (P-Smad2) (1:400; bs-8853R, Bioss, Beijing, China), phosphorylated Smad3 (P-Smad3) (1:400; bs-3425R, Bioss, Beijing, China), BMPR2 (1:500, bs-4237R; Bioss, Beijing, China), Smad1/5 (1:500; AF0614, Affinity, Jiangsu, China), phosphorylated Smad1/5 (P-Smad1/5) (1:400; bs-3418R, Bioss, Beijing, China), PCNA (1:500; bs-2006R, Bioss, Beijing, China), and BCL-2 (1:500; bs-0032R, Bioss, Beijing, China).

Techniques: Expressing, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

doi: 10.1161/JAHA.118.009244

Figure Lengend Snippet: Efficiency of microRNA‐155 (miR‐155) inhibition and overexpression. A, Diagram describing the experimental setup. Human primary brain microvascular endothelial cells (HBMECs) seeded in cell culture inserts were subjected to 3 hours of oxygen‐glucose deprivation (OGD) and returned back to the normal cell culture conditions; 24 hours later, the cells were transfected with miR‐155 inhibitor, mimic, or appropriate scrambled oligonucleotide. Cells were analyzed at 48 hours after the transfection. B, Fluorescence confocal microscopy of HBMEC s transfected with fluorescein‐labeled miR‐155 inhibitor control (green dots) and stained for actin with rhodamine‐conjugated fluorescent phalloidin. Left panel: orthogonal image projection verifies that fluorescent probes were incorporated within the cell. Bar: 10 μm. C and D, miR‐155 PCR analysis. Total RNA was isolated from the cells subjected to OGD and transfected with the following oligonucleotides: miR‐155 mimic ( OGD /M; grey bar); mimic control ( OGD / MC; grey bar with white stripes); specific miR‐155 inhibitor ( OGD /I; black bar); and control inhibitor ( OGD / IC ; black bar with white stripes). C, P =0.002, (D) P =0.029, Mann–Whitney (Wilcoxon) test. n=3 (for OGD / MC and OGD / IC groups) and n=4 (for OGD /I and OGD /M groups) independent experiments. E, Validation of miR‐155 inhibition by the quantitative assessment of miR‐155 direct target proteins Rictor and SMAD ‐1. Optical density of the protein bands was measured using ImageJ software, normalized to GAPDH density in every sample, and expressed as average relative density values. Protein levels were compared between OGD /I (black bars) and OGD / IC (black bars with white stripes) cell lysates. P =0.029, Mann–Whitney (Wilcoxon) test, n=3 independent experiments per group. Representative immunoblots demonstrate Rictor and SMAD ‐1 protein expression (3 bands per group); GAPDH was used as a loading control. Error bars: SEM ; * P <0.05; ** P <0.01.

Article Snippet: SMAD‐1 and Rictor proteins were detected using SMAD proteins (Cat# 12656) and mTOR pathway (Cat# 9964) antibody sampler kits (Cell Signaling).

Techniques: Inhibition, Over Expression, Cell Culture, Transfection, Fluorescence, Confocal Microscopy, Labeling, Control, Staining, Isolation, MANN-WHITNEY, Biomarker Discovery, Software, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Graphical representation of the experimental design. Experiment 1: Changes in pain sensitization and expressions of BMP10, ALK2, Smad1/5/8, phosphorylated Smad1/5/8, and GFAP after SNI in mice. Experiment 2: The effects of BMP10 siRNA on SNI-induced pain hypersensitivity and astrocytic activation. Experiment 3: The involvements of ALK2 in BMP10-induced pain hypersensitivity and astrocytic activation. Experiment 4: The effects of Smad1 siRNA on BMP10-induced pain hypersensitivity and astrocytic activation.

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in mouse ipsilateral spinal dorsal horn after SNI. (A–C) Western blot analysis showed the expression levels of ALK2, Samd1/5/8, and p-Smad1/5/8 in the ipsilateral (above) and contralateral (below) spinal dorsal horn of sham and SNI mice; (D) Double immunofluorescence staining showed the coexpression of ALK2 (red) with GFAP, Iba-1 and NeuN (green) in the ipsilateral spinal dorsal horn of SNI mice on postoperative Day 14 (scale bar = 50 μm/25 μm). Data are presented as mean and SEM; sham mice versus SNI mice on postoperative Days 7 and 14, *** p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Double Immunofluorescence Staining

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Smad1 was pivotal in astrocytic activation in vitro . (A) qRT-PCR analysis showed the expression levels of Smad1 mRNA in cultured astrocytes transfected with siRNAs; (B) Western blot analysis showed the expression levels of Smad1 protein in cultured astrocytes transfected with siRNAs data are presented as mean and SEM, Smad1 siRNA group versus + Control group *** p < 0.001 (n = 6); (C) The purity of cultured astrocytes (scale bar = 200 μm); (D) The CCK assay showed the cell viability of cultured astrocytes; (E, F) Immunofluorescence staining results showed the expression of GFAP in cultured astrocytes transfected with siRNAs (scale bar = 50 μm); (G, H) Western blot showed the expressions of GFAP and p-Smad1/5/8 and in cultured cells; data are presented as mean and SEM, NC siRNA + Vehicle group versus NC siRNA + LPS group *** p < 0.001; NC siRNA + LPS group versus Smad1 siRNA + LPS group ### p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay, In Vitro, Quantitative RT-PCR, Expressing, Cell Culture, Transfection, Western Blot, Control, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Intrathecal injection of Smad1 siRNA knocked down the expression of Smad1 without obvious side effects. (A) qRT-PCR analysis showed the expression levels of Smad1 mRNA in the spinal cord of mice after intrathecal injection with vehicle, NC siRNA, Smad1 siRNA; (B, C) Western blot analysis showed the expression levels of Smad1 protein in the spinal cord of mice after intrathecal injected with vehicle, NC siRNA, Smad1 siRNA; (D, E) Paw withdrawal threshold (PWT) and Thermal withdrawal latency (TWL) of mice after intrathecal injected with vehicle, NC siRNA, Smad1 siRNA (F) Representative movement traces in OFT tests of mice after intrathecal injected with vehicle, NC siRNA, Smad1 siRNA (G–I) Time in center zone, average speed and total distance of mice after intrathecal injected with vehicle, NC siRNA, Smad1 siRNA. Data were presented as mean and SEM, Smad1 siRNA group versus Control group *** p < 0.001; Smad1 siRNA group versus NC siRNA group ### p < 0.001, n = 6.

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Injection, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: BMP10 accelerated spinal astrocytic activation in neuropathic pain via ALK2/smad1/5/8 signaling

doi: 10.3389/fphar.2024.1426121

Figure Lengend Snippet: Smad1 was involved in BMP10-induced pain hypersensitivity and astrocytic activation. (A, B) PWT and TWL of normal mice after intrathecal delivery of siRNA and BMP10 peptide; (C, D) Western blot showed the expressions of p-Smad1/5/8 and GFAP in the spinal cord of normal mice after intrathecal delivery of siRNA and BMP10 peptide; (E, F) immunofluorescence staining showed the expression of GFAP in the spinal dorsal horn of normal mice after the intrathecal delivery of siRNA and BMP10 peptide; (scale bar = 200 μm/50 μm); Data are presented as mean and SEM, NC siRNA + Vehicle group versus NC siRNA + BMP10 group *** p < 0.001; NC siRNA + BMP10 group versus Smad1 siRNA + BMP10 group # p < 0.05, ### p < 0.001 (n = 6).

Article Snippet: The membranes were incubated with appropriate antibodies, including mouse anti-GFAP antibody (1:1,000, ab279289, Abcam), mouse anti-BMP10 antibody (1:1,000, 462732, Novus Biologicals, CO, United States), rabbit anti-ALK2 antibody (1:100, PA5-114818, Thermo Fisher Scientific, MA, United States), rabbit anti-phospho-Smad1/5/8 (1:1,000, AB3848-I, Sigma Aldrich, Germany), rabbit anti-Smad1/5/8 (1:1,000, NB100-56656, Novus Biologicals, CO, United States) and mouse anti-β-actin (1:1,000, AF2815, Beyotime Biotechnology , Shanghai, China) overnight at 4°C.

Techniques: Activation Assay, Western Blot, Immunofluorescence, Staining, Expressing