Journal: PLoS ONE

Article Title: Nicotine Stimulates Nerve Growth Factor in Lung Fibroblasts through an NFκB-Dependent Mechanism

doi: 10.1371/journal.pone.0109602

Figure Lengend Snippet: Using a transwell system, primary murine lung fibroblasts were cultured on an upper chamber transwell membrane and primary murine ASM cells were cultured on the lower plastic chamber. Both chambers were cultured in serum free medium. The upper chamber containing fibroblasts was treated with of nicotine (50 µg/ml) for 72 hours. Primary murine ASM cells were harvested for protein isolation. Phosphorylated (p-MLC) and total myosin light chain (total MLC) expression was measured by using immunoblot analysis with densitometry. ASM cells cultured in proximity to nicotine treated fibroblasts ( N ) have significantly increased higher p-MLC/total MLC ratio compared to controls ( C ). n = 6, *p<0.05, error bars represent ±SEM.

Article Snippet: Human ASM cells were purchased through Lonza (Portsmouth, NH) and cultured according to the manufacturer’s protocol.

Techniques: Cell Culture, Membrane, Isolation, Expressing, Western Blot

Journal:

Article Title: The orphan nuclear receptor ROR? is a negative regulator of the inflammatory response

doi: 10.1093/embo-reports/kve007

Figure Lengend Snippet: Fig. 1. RORα is expressed in different vascular SMC types. (A and B) RT–PCR (35 cycles) analysis of RORα and GAPDH mRNA. C-PCR and C-RT are negative controls for PCR and RT, respectively; VSMC, smooth muscle cells from saphenous veins; CASMC, human coronary artery smooth muscle cells; HASMC, human aortic smooth muscle cells. (C) RT–PCR (30 cycles) analysis of RORα mRNA in SMC infected with Ad-RORα1 or Ad-GFP for 24 h. (D) Analysis of RORα protein expression in SMC infected for 24 h with or without Ad-RORα1. Immunocytochemistry experiments were performed as previously described (Chinetti et al., 1998) using a rabbit polyclonal RORα antibody raised against aa 163–225.

Article Snippet: Primary human aortic (HA) and coronary artery (CA) SMC (PromoCell, Heidelberg, Germany) and primary SMC from saphenous veins (VSMC: a kind gift of Dr Walsh, Boston, MA) were cultured under standard conditions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Immunocytochemistry

Journal: International Journal of Biological Sciences

Article Title: Irisin attenuates vascular remodeling in hypertensive mice induced by Ang II by suppressing Ca 2+ -dependent endoplasmic reticulum stress in VSMCs

doi: 10.7150/ijbs.84153

Figure Lengend Snippet: Irisin supplementation suppressed the proliferation, migration and phenotypic transformation in Ang II-challenged VSMCs. A. The cell viability in cultured primary mouse VSMCs challenged by Ang II (1 μM) in the absence and presence of exogenous irisin (5, 10, 20 nM) co-incubation for 24 h (n=4/group). B. Edu incorporation assays evaluated the proliferation ability of VSMCs challenged by Ang II (1 μM) in the absence and presence of exogenous irisin (20 nM) co-incubation for 24 h (n=4/group). C. Scratch and transwell migration assays evaluated the migration of VSMCs challenged by Ang II (1 μM) in the absence and presence of exogenous irisin (20 nM) co-incubation for 24 h (n=4/group). D. Phenotypic switching indicated by the mRNA expressions of VSMC-specific contractile genes (including SMA, SM22, calponin, and caldesmon1) in VSMCs challenged by Ang II (1 μM) in the absence and presence of exogenous irisin (20 nM) co-incubation for 24 h (n=5/group). The data were presented as mean ± S.E.M., and the statistical analyses were performed by one way ANOVA. *P < 0.05 and **P < 0.01. Ctrl, control; Ang II, angiotensin II; SMA, SM α-actin; SM22, smooth muscle 22 alpha.

Article Snippet: Subsequently, the sections were incubated overnight in darkness at 4°C with a mouse anti-Ki-67 primary antibody (1:100) and an anti-SMC-α-actin antibody (1:150) from Cell Signaling Technology.

Techniques: Migration, Transformation Assay, Cell Culture, Incubation, Control

Journal: International Journal of Biological Sciences

Article Title: Irisin attenuates vascular remodeling in hypertensive mice induced by Ang II by suppressing Ca 2+ -dependent endoplasmic reticulum stress in VSMCs

doi: 10.7150/ijbs.84153

Figure Lengend Snippet: Irisin supplementation alleviated intracellular calcium imbalance-mediated ER stress induced by Ang II exposure. A. Ca 2+ fluorescent probe assays evaluated intracellular calcium levels in VSMCs challenged with Ang II (1 μM) or irisin (20 nM) alone, or irisin (20 nM) or YM-58483 (a SOCE inhibitor, 10 μM) pre-treated for 30 min, and then co-incubation with Ang Ⅱ (1 μM) for another 6 h (n=4/group). B. The effect of irisin supplementation (20 nM) on the expression levels of calcium-signaling related proteins in VSMCs with Ang II exposure (1 μM, n=3/group). C. The effect of irisin (20 nM) or BAPTA-AM (a Ca 2+ chelator, 1 μM) supplementation on the expression of ER stress-related proteins in VSMCs with Ang II (1 μM) exposure (n=3/group). D. Edu incorporation assays evaluated the proliferation ability of VSMCs treated with Ang II (1 μM), or 4-PBA (an ERS inhibitor, 1mM), or BAPTA-AM (a Ca 2+ chelator, 1 μM) alone, or Ang II (1 μM) in combination with 4-PBA (1mM) or BAPTA-AM (1 μM) for 24 h, respectively (n=4/group). E. Transwell migration assays evaluated the migration of VSMCs treated with Ang II (1 μM), or 4-PBA (an ERS inhibitor, 1mM), or BAPTA-AM (a Ca 2+ chelator, 1 μM) alone, or Ang II (1 μM) in combination with 4-PBA (1mM) or BAPTA-AM (1 μM) for 24 h, respectively (n=4/group). F. Phenotypic switching indicated by the mRNA expressions of VSMC-specific contractile-genes (including SMA, SM22, calponin, and caldesmon1) in VSMCs treated with Ang II (1 μM), or 4-PBA (an ERS inhibitor, 1mM), or BAPTA-AM (a Ca 2+ chelator, 1 μM) alone, or Ang II (1 μM) in combination with 4-PBA (1mM) or BAPTA-AM (1 μM) for 24 h, respectively (n=4/group). The data were presented as mean ± S.E.M., and the statistical analyses were performed by one way ANOVA. *P < 0.05 and **P < 0.01. Ctrl, control; Ang II, angiotensin II; SMA, SM α-actin; SM22, smooth muscle 22 alpha.

Article Snippet: Subsequently, the sections were incubated overnight in darkness at 4°C with a mouse anti-Ki-67 primary antibody (1:100) and an anti-SMC-α-actin antibody (1:150) from Cell Signaling Technology.

Techniques: Incubation, Expressing, Migration, Control

Journal: International Journal of Biological Sciences

Article Title: Irisin attenuates vascular remodeling in hypertensive mice induced by Ang II by suppressing Ca 2+ -dependent endoplasmic reticulum stress in VSMCs

doi: 10.7150/ijbs.84153

Figure Lengend Snippet: Irisin inhibited calcium overload, ER stress and remodeling in VSMCs and aortic tissues with Ang II exposure via αV/β5 receptor-AMPK/p38 pathway. A and B. The effect of irisin supplementation on the expression levels of ERS-related proteins and kinases (AMPK and p38) in Ang II-treated VSMCs was evaluated by immunoblotting. VSMCs were treated with irisin (20 nM) or Ang II (1 μM) alone, or irisin (20 nM) in combination with Ang II (1 μM) in the absence and presence of αV/β5 antibody (n=3/group). C. Intracellular calcium levels in VSMCs treated with irisin (20 nM) or Ang II (1 μM) alone, or irisin (20 nM) in combination with Ang II (1 μM) in the absence and presence of αV/β5 antibody (n=3/group). D-E. The levels of phosphorylated- and total-AMPK and p38 in lysates of aortic tissues from WT, irisin-KO, and irisin-OV mice infused with saline or Ang II (490 ng/min/kg) for 4 weeks were detected using immunoblotting (n=3/group). F. Edu incorporation assay evaluated the proliferation ability of VSMCs treated with irisin (20 nM) or Ang II (1 μM) alone, or irisin (20 nM) in combination with Ang II (1 μM) in the absence and presence of αV/β5 antibody, or compound C (an AMPK inhibitor, 5 μM), or HY-W007355 (a p38 activator, 10 μM) for 24 h (n=4/group). G. Transwell migration assay evaluated the migration of VSMCs treated with irisin (20 nM) or Ang II (1 μM) alone, or irisin (20 nM) in combination with Ang II (1 μM) in the absence and presence of αV/β5 antibody, or compound C (an AMPK inhibitor, 5 μM), or HY-W007355 (a p38 activator, 10 μM) for 24 h (n=4/group). H. Phenotypic switching indicated by the mRNA expressions of VSMC-specific contractile genes (including SMA, SM22, calponin, and caldesmon1) in VSMCs treated with irisin (20 nM) or Ang II (1 μM) alone, or irisin (20 nM) in combination with Ang II (1 μM) in the absence and presence of αV/β5 antibody, or compound C (an AMPK inhibitor, 5 μM), or HY-W007355 (a p38 activator, 10 μM) for 24 h (n=4/group). The data were presented as mean ± S.E.M., and the statistical analyses were performed by one way ANOVA. *P < 0.05 and **P < 0.01. Ctrl, control; I, irisin; Ang II, angiotensin II; SMA, SM α-actin; SM22, smooth muscle 22 alpha; WT, wild type mice; KO, irisin knockout mice; OV, irisin overexpression mice.

Article Snippet: Subsequently, the sections were incubated overnight in darkness at 4°C with a mouse anti-Ki-67 primary antibody (1:100) and an anti-SMC-α-actin antibody (1:150) from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Saline, Transwell Migration Assay, Migration, Control, Knock-Out, Over Expression

Journal: The Journal of allergy and clinical immunology

Article Title: A functional splicing variant associated with decreased asthma risk abolishes the ability of gasdermin B ( GSMDB ) to induce epithelial cell pyroptosis

doi: 10.1016/j.jaci.2017.11.040

Figure Lengend Snippet: A) Relative GSDMB mRNA expression in human primary airway smooth muscle (ASM), lung fibroblasts (FIB), and normal human bronchial epithelial (NHBE) cells over the course of air-liquid interface (ALI) culture. ACTB (β-actin) was used as an internal control. B) Relative GSDMB mRNA expression in sorted β−tubulin IV-positive ciliated NHBE and MUC5AC-postive goblet NHBE cells. Graphs show mean of fold-change from three different donors; n=3, +/− SEM. C and D) Immunostaining of GSDMB protein in NHBE (normal human bronchial epithelial) cells at day 21 of ALI culture (C) and lung tissue (D) shows GSDMB expression in ciliated cells. Ciliated cells were stained for β−tubulin IV and nuclei were visualized by DAPI staining.

Article Snippet: Primary human airway smooth muscle (ASM) cells and normal human lung fibroblasts were obtained from Lonza.

Techniques: Expressing, Control, Immunostaining, Staining