human asm cells  (Lonza)

Journal: Bioengineered

Article Title: MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1

doi: 10.1080/21655979.2022.2036890

Figure Lengend Snippet: The effects of TNF-α on miR-15b-5p expression in ASM cells.

Article Snippet: Human primary ASM cells (ATCC PCS-130-011TM, Manassas, VA, USA) were subsequently cultured in Dulbecco’s modified Eagles medium/Ham’s F12 (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 unit/mL penicillin, and 100 μg/mL streptomycin (Cellgro, Manassas, VA, USA).

Techniques: Expressing

Journal: Bioengineered

Article Title: MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1

doi: 10.1080/21655979.2022.2036890

Figure Lengend Snippet: The effects of miR-15b-5p on TNF-α-induced proliferation and migration of ASM cells.

Article Snippet: Human primary ASM cells (ATCC PCS-130-011TM, Manassas, VA, USA) were subsequently cultured in Dulbecco’s modified Eagles medium/Ham’s F12 (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 unit/mL penicillin, and 100 μg/mL streptomycin (Cellgro, Manassas, VA, USA).

Techniques: Migration

Journal: Bioengineered

Article Title: MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1

doi: 10.1080/21655979.2022.2036890

Figure Lengend Snippet: The effects of miR-15b-5p on TNF-α-induced inflammatory response and ECM deposition in ASM cells.

Article Snippet: Human primary ASM cells (ATCC PCS-130-011TM, Manassas, VA, USA) were subsequently cultured in Dulbecco’s modified Eagles medium/Ham’s F12 (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 unit/mL penicillin, and 100 μg/mL streptomycin (Cellgro, Manassas, VA, USA).

Techniques:

Journal: Bioengineered

Article Title: MicroRNA-15b-5p inhibits tumor necrosis factor alpha-induced proliferation, migration, and extracellular matrix production of airway smooth muscle cells via targeting yes-associated protein 1

doi: 10.1080/21655979.2022.2036890

Figure Lengend Snippet: The restoration of YAP1 expression reverses the effect of the upregulated miR-15b-5p on ASM cells.

Article Snippet: Human primary ASM cells (ATCC PCS-130-011TM, Manassas, VA, USA) were subsequently cultured in Dulbecco’s modified Eagles medium/Ham’s F12 (Hyclone, Logan, UT, USA) with 10% fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 100 unit/mL penicillin, and 100 μg/mL streptomycin (Cellgro, Manassas, VA, USA).

Techniques: Expressing

Journal: Respiratory research

Article Title: Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

doi: 10.1186/s12931-023-02465-8

Figure Lengend Snippet: Fig. 1 DGK inhibitor attenuation of ASM cell proliferation involves PKA. GFP- and PKI-GFP-expressing human ASM cells were pretreated with DGK I (0–20 µM) followed by PDGF (10 ng/ml) stimulation for 48 h and cell proliferation was measured by assessing total DNA. Graphical representation is of mean ± SEM (n = 6 distinct donors) of CyQuant fluorescence normalized to basal (A). Expression of GFP in ASM cells transduced with retroviral particles was confirmed by fluorescence microscopy. Representative florescence (top) and brightfield (bottom) images of two different lines are shown (B). *p < 0.05 (compared to matched basal) and #p < 0.05 (compared to matched PDGF) using one-way-ANOVA with Bonferroni post-hoc analysis

Article Snippet: Human ASM cells were plated in 12-well plates and maintained in HAM’s F-12 + 10% FBS media for 24 h. Cells were then serum-starved with HAM’s F-12 medium containing 1% ITS for 24 h followed by pre-treatment with vehicle or pan-PKC (Bis I), MEK (U0126) or ERK2 (Vx11e) inhibitor for 10 min, and then stimulated with vehicle or DGK I (20 μM) for 24 h. In a subset of experiments, cells were stimulated with vehicle or DGK I (20 μM) for 0, 5, 10, 15 and 30 min, or 24 h. Human ASM cells were lysed in RIPA buffer (CST, USA) supplemented with protease and phosphatase inhibitor (Bimake) at 4 °C for 30 min. Lysates were then mixed with Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol and boiled at 95 °C for 5 min.

Techniques: Expressing, CyQUANT Assay, Fluorescence, Transduction, Retroviral, Microscopy

Journal: Respiratory research

Article Title: Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

doi: 10.1186/s12931-023-02465-8

Figure Lengend Snippet: Fig. 2 DGK inhibition enhances COXII expression and PGE2 production. Human ASM cells were treated with DGK I (20 µM) in a time-dependent manner. Cells were lysed and immunoblotted for COXII and β-actin (A). Graphical representation of mean ± SEM of COXII densitometry data normalized to basal conditions (B). Supernatant collected was subjected to ELISA to assay amounts of PGE2 (pg/ml) secreted and data were normalized to vehicle-treated condition (C). Data above are mean ± SEM (n = 3–4 distinct donors). *p < 0.05 (DMSO vs. DGK I at matched time points) using one-way-ANOVA with Bonferroni post-hoc analysis. Please note that the gel images shown are cropped from the full length images

Article Snippet: Human ASM cells were plated in 12-well plates and maintained in HAM’s F-12 + 10% FBS media for 24 h. Cells were then serum-starved with HAM’s F-12 medium containing 1% ITS for 24 h followed by pre-treatment with vehicle or pan-PKC (Bis I), MEK (U0126) or ERK2 (Vx11e) inhibitor for 10 min, and then stimulated with vehicle or DGK I (20 μM) for 24 h. In a subset of experiments, cells were stimulated with vehicle or DGK I (20 μM) for 0, 5, 10, 15 and 30 min, or 24 h. Human ASM cells were lysed in RIPA buffer (CST, USA) supplemented with protease and phosphatase inhibitor (Bimake) at 4 °C for 30 min. Lysates were then mixed with Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol and boiled at 95 °C for 5 min.

Techniques: Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Respiratory research

Article Title: Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

doi: 10.1186/s12931-023-02465-8

Figure Lengend Snippet: Fig. 3 DGK inhibitor-induced COXII production and PKA activation is mediated via PKC-ERK1/2 signaling. Human ASM cells were pretreated with vehicle, Bis I (10 µM), U0126 (1 µM), or Vx11e (1 µM) for 10 min followed by stimulation with vehicle or DGK I (20 µM) for 24 h. Cells were lysed and immunoblotted for COXII, p-CREB, VASP, and β-actin (A). Graphical representation of mean ± SEM (n = 3–5 distinct donors) for COXII (B), p-CREB (C) and VASP (D). The densitometry data for COXII and p-CREB are normalized to basal conditions. Densitometry data for VASP phosphorylation are presented as percent (%) VASP shift (p-VASP/total VASP). *p < 0.05 (compared to vehicle basal) and #p < 0.05 (compared to DGK I basal) using one-way-ANOVA with Bonferroni post-hoc analysis. Please note that the gel images shown are cropped from the full length images

Article Snippet: Human ASM cells were plated in 12-well plates and maintained in HAM’s F-12 + 10% FBS media for 24 h. Cells were then serum-starved with HAM’s F-12 medium containing 1% ITS for 24 h followed by pre-treatment with vehicle or pan-PKC (Bis I), MEK (U0126) or ERK2 (Vx11e) inhibitor for 10 min, and then stimulated with vehicle or DGK I (20 μM) for 24 h. In a subset of experiments, cells were stimulated with vehicle or DGK I (20 μM) for 0, 5, 10, 15 and 30 min, or 24 h. Human ASM cells were lysed in RIPA buffer (CST, USA) supplemented with protease and phosphatase inhibitor (Bimake) at 4 °C for 30 min. Lysates were then mixed with Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol and boiled at 95 °C for 5 min.

Techniques: Activation Assay, Phospho-proteomics

Journal: Respiratory research

Article Title: Crosstalk between diacylglycerol kinase and protein kinase A in the regulation of airway smooth muscle cell proliferation.

doi: 10.1186/s12931-023-02465-8

Figure Lengend Snippet: Fig. 4 DGK inhibition activates ERK1/2 signaling in a time-dependent manner. Human ASM cells were treated with vehicle or DGK I (20 µM) for 0, 5, 10, 15 and 30 min. Cells were lysed and immunoblotted for ERK1/2 phosphorylation and β-actin (A). Graphical representation of mean ± SEM (n = 3 distinct donors) for ERK1/2 phosphorylation (B). The densitometry data for ERK1/2 phosphorylation by DGK I were normalized to matched vehicle-treated controls at each time point, and graphed as fold change from the basal. *p < 0.05 (compared to vehicle basal) using one-way-ANOVA with Bonferroni post-hoc analysis. Please note that the gel images shown are cropped from the full length images

Article Snippet: Human ASM cells were plated in 12-well plates and maintained in HAM’s F-12 + 10% FBS media for 24 h. Cells were then serum-starved with HAM’s F-12 medium containing 1% ITS for 24 h followed by pre-treatment with vehicle or pan-PKC (Bis I), MEK (U0126) or ERK2 (Vx11e) inhibitor for 10 min, and then stimulated with vehicle or DGK I (20 μM) for 24 h. In a subset of experiments, cells were stimulated with vehicle or DGK I (20 μM) for 0, 5, 10, 15 and 30 min, or 24 h. Human ASM cells were lysed in RIPA buffer (CST, USA) supplemented with protease and phosphatase inhibitor (Bimake) at 4 °C for 30 min. Lysates were then mixed with Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol and boiled at 95 °C for 5 min.

Techniques: Inhibition, Phospho-proteomics

Journal: ACS nano

Article Title: Molecular nanomechanical mapping of histamine-induced smooth muscle cell contraction and shortening

doi: 10.1021/acsnano.1c01782

Figure Lengend Snippet: (A) Representative ASM cell morphologies (DIC) before and after histamine injection on fibronectin, biotinylated RGD, 54 pN TGT and 43 pN TGT coated surfaces. Scale, 10 μm.

Article Snippet: Immortalized human ASM cell line (Normal12 human ASM cell line) was purchased from Discovery Biomed, Inc. (Birmingham, AL) and cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and F12 (Corning, 15–090-CV) supplemented with 1X Insulin-Transferrin-Selenium (ThermoFisher, 41400045), 1X Antibiotic-Antimycotic (Gibco, 15240062), and 10% fetal bovine serum in 5% CO 2 at 37 °C.

Techniques: Injection

Journal: ACS nano

Article Title: Molecular nanomechanical mapping of histamine-induced smooth muscle cell contraction and shortening

doi: 10.1021/acsnano.1c01782

Figure Lengend Snippet: Histamine induced ASM cell shortening on multiplexed qTGT surface (12 pN qTGT with BHQ2-Cy3 and 54 pN qTGT with BHQ2-Atto647N). Cell shortening was captured after the treatment of histamine (100 μM).

Article Snippet: Immortalized human ASM cell line (Normal12 human ASM cell line) was purchased from Discovery Biomed, Inc. (Birmingham, AL) and cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium and F12 (Corning, 15–090-CV) supplemented with 1X Insulin-Transferrin-Selenium (ThermoFisher, 41400045), 1X Antibiotic-Antimycotic (Gibco, 15240062), and 10% fetal bovine serum in 5% CO 2 at 37 °C.

Techniques:

Journal: Science signaling

Article Title: Airway relaxation mechanisms and structural basis of osthole for improving lung function in asthma

doi: 10.1126/scisignal.aax0273

Figure Lengend Snippet: (A and B) Human ASM cells were transfected without (mock) or with scrambled or PDE4D siRNA. Cells were subjected to quantitative RT-PCR analysis of relative PDE4D mRNA expression (A) and Western blot analysis of VASP phosphorylation stimulated without (control) or with osthole (3 μM) or IBMX (500 μM) for 15 min (B). (C) VASP phosphorylation in human ASM cells stimulated without (control) or with 3 μM osthole, 10 μM GEBR-7, or the combination of drugs. The data are means ± SEM (n = 3 different ASM cell lines). (D) Relaxation of 0.3 μM ACh–preconstricted airways induced by vehicle (control), 10 μM osthole, 25 μM GEBR-7, or the combination of drugs in mouse PCLS. The data (means ± SEM) were generated in at least five PCLS from three mice for each group. (E to G) Mouse airways were preconstricted with MCh (150 mg/ml). Ten microliters of osthole (2.5 mg/ml) before (E) or after GEBR-7b (2.5 mg/ml) (F) was sequentially aerosolized over 30 s to assess bronchodilator rescue as indicated by the average decrease in lung resistance (RL) 2 min after aerosol administration. (G) Representative time course of bronchodilator rescue of Alb (10 μl, 2.5 mg/ml) aerosolized into mouse lungs after osthole or GEBR-7b. N = 4 mice for each group in each experiment. **P < 0.01 using the Kruskal-Wallis test (A); **P < 0.01 using ANOVA with the Bonferroni correction for multiple comparisons test (B to F) and N.S. for nonsignificance (E and F). KD, knockdown.

Article Snippet: Knockdown of PDE4D in human ASM cells by dual transfection of siRNA Human ASM cells (1.2 × 10 5 ) were transfected without (mock) or with PDE4D siRNA (si-h-PDE4D_101) or scramble siRNA (siR NC #1) (RiboBio) using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Phospho-proteomics, Control, Generated, Aerosol, Knockdown