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

Article Title: Smad3 Regulates Neuropilin 2 Transcription by Binding to its 5′ Untranslated Region

doi: 10.1161/JAHA.119.015487

Figure Lengend Snippet: Smad3 regulates neuropilin 2 (NRP2) expression in cultured smooth muscle cells (SMCs). A–B , Smad3 loss‐of‐function reduces NRP2 expression levels. C–D , Smad3 gain‐of‐function increases NRP2 expression levels. Human primary aortic SMCs were transfected with scrambled small interfering RNA, Smad3‐specific small interfering RNA, empty vector, or Smad3 overexpression plasmid for 12 hours in basal medium (no fetal bovine serum). The cells were cultured for another 12 hours in fresh basal medium (no Lipofectamine) to recover, and then treated with 10 ng/mL of transforming growth factor β for 20 hours before harvest for Western blot and quantitative real‐time polymerase chain reaction (qRT‐PCR) analyses. Quantification: densitometry of Western blots (similar enhanced chemiluminescent exposure) from independent repeat experiments was normalized (to GAPDH) and then averaged to calculate mean±SEM (n=3). Readings of triplicate qRT‐PCR reactions were normalized (to GAPDH) and averaged to calculate mean±SD (n=3). Statistics: 1‐way ANOVA followed by Bonferroni post hoc test; *** P <0.001. TSS indicates transcriptional start site; and WT wild‐type.

Article Snippet: Human aortic SMCs (AoSMCs, CC‐2571), SMC basal medium (CC‐3181), and SMC basal medium plus SingleQuots of supplements (CC‐3182) were purchased from Lonza.

Techniques: Expressing, Cell Culture, Transfection, Small Interfering RNA, Plasmid Preparation, Over Expression, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

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

Article Title: Smad3 Regulates Neuropilin 2 Transcription by Binding to its 5′ Untranslated Region

doi: 10.1161/JAHA.119.015487

Figure Lengend Snippet: While silencing Smad3 mitigates pro–intimal hyperplasia (IH) smooth muscle cell (SMC) phenotypes, neuropilin 2 (NRP2) silencing abolishes Smad3’s enhancement of these phenotypes. A–C , Smad3 silencing mitigates pro‐IH SMC phenotypes. D–G , NRP2 silencing abolishes Smad3 overexpression (Smad3‐OE)–enhanced pro‐IH SMC phenotypes. For loss‐ or gain‐of‐function, transfection of human aortic SMCs was performed as described for Figure . In proliferation experiments, transfected cells were treated with transforming growth factor β (TGFβ1) (10 ng/mL) for 48 hours before CellTiter‐Glo viability assay. We started with more cells in ( D ) (vs A), considering 2‐step manipulations (transfection for Smad3 expression followed by that for NRP2 silencing). To monitor migration, transfected cells were seeded in the Transwell insert, the lower chamber filled with full medium (and 10 ng/mL TGFβ1). Cells that migrated to the lower surface of the insert were imaged after 24 hours of incubation. To determine SMC dedifferentiation, transfected cells were induced with cholesterol (10 ng/mL) for 72 hours in basal medium and then harvested for Western blot determination of α‐smooth muscle actin and CD68 protein levels. Quantification (mean±SEM, n=3) was performed as described for Figure . Statistics: 1‐way ANOVA followed by Bonferroni post hoc test; * P <0.05, ** P <0.01, *** P <0.001. Scramble indicates scrambled small interfering RNA.

Article Snippet: Human aortic SMCs (AoSMCs, CC‐2571), SMC basal medium (CC‐3181), and SMC basal medium plus SingleQuots of supplements (CC‐3182) were purchased from Lonza.

Techniques: Over Expression, Transfection, Viability Assay, Expressing, Migration, Incubation, Western Blot, Small Interfering RNA

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