Journal: Advanced healthcare materials

Article Title: Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step Towards Personalized Mechano-medicine

doi: 10.1002/adhm.201800069

Figure Lengend Snippet: Measuring airway smooth muscle (ASM) cell integrin forces using titin-based molecular tension probes. A) Schematic illustration of the RGD-Cy3-I27 MTFM sensor and its mechanism of reporting integrin forces. B) Representative AFM image of 9 nm AuNPs immobilized on a PEGylated [5% (w/v) mPEG-NHS and 0.5% (w/v) lipoic acid-PEG-NHS] glass substrate. Scale bar, 200 nm. C–D) Representative RICM and fluorescence images of human ASM cells on the tension sensing substrate before and after treatment with ROCK kinase inhibitor Y-27632 (40 μM) or ML-7 (40 μM) for 30 min. Scale bar, 10 μm. E) Plot shows the normalized tension signal, defined as Idrug/Iuntreated, of the same cells (same ROI) before and after ROCK inhibitor and ML-7 treatment. Masks were created to isolate tension signal from background. Lines represent mean±SEM from n = 9 cells for Y-27632, and n = 5 cells for ML-7 treatment from three independent surface preparations, **P <0.01 (Y-27632) and ***P < 0.001 (ML-7) by Wilcoxon signed-rank test. F) Representative RICM and fluorescence images of human ASM cells transfected with GFP-vinculin incubated on the tension sensor before and after treatment with ROCK kinase inhibitor Y-27632 (40 μM) for 3 min. Scale bar, 10 μm.

Article Snippet: ASM cells were transiently transfected with either GFP-actin, GFP-vinculin or GFP-paxillin using Lipofectamine® LTX with Plus ™ reagent by mixing 1 μg of DNA with Lipofectamine® LTX and Plus ™ Reagent for each well in a 24-well plate and incubated for 24–48 h. Western blot After overnight serum starvation, human ASM cells (Lonza, Switzerland) ± nicotine (50 μg/ml, 72 h) and human ASM cells isolated from healthy and asthmatic donors were cultured in serum-free media.

Techniques: Fluorescence, Transfection, Incubation

Journal: Advanced healthcare materials

Article Title: Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step Towards Personalized Mechano-medicine

doi: 10.1002/adhm.201800069

Figure Lengend Snippet: Integrin mediated forces are enhanced in the asthmatic human ASM cells and in the presence of nicotine. A) Representative RICM and integrin-mediated tension images of normal and asthmatic human ASM cells incubated on the RGD-Cy3-I27 tension probe for 2 h. Scale bar, 10 μm. B) Representative RICM and tension images of normal and asthmatic ASM cells treated for 72 h with nicotine (50 μg/ml), for 24 h with doxycycline (4 μg/ml) and incubated on the titin-based tension probe for 2 h. Scale bar, 10 μm. C) Scatter plot quantifying the integrated fluorescence intensity of FAs from normal and asthmatic donors with and without addition of nicotine for 72 h. Each circle represents data collected from a single cell. The red line indicates the mean, while the box shows the SEM, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using Student’s t-test. D) Scatter plot showing the integrated fluorescence intensity of FAs pooled from 3 normal and 4 asthmatic donors with and without addition of nicotine for 72 h. ****P<0.0001 using Mann-Whitney test.

Article Snippet: ASM cells were transiently transfected with either GFP-actin, GFP-vinculin or GFP-paxillin using Lipofectamine® LTX with Plus ™ reagent by mixing 1 μg of DNA with Lipofectamine® LTX and Plus ™ Reagent for each well in a 24-well plate and incubated for 24–48 h. Western blot After overnight serum starvation, human ASM cells (Lonza, Switzerland) ± nicotine (50 μg/ml, 72 h) and human ASM cells isolated from healthy and asthmatic donors were cultured in serum-free media.

Techniques: Incubation, Fluorescence, MANN-WHITNEY

Journal: Advanced healthcare materials

Article Title: Molecular Tension Probes to Investigate the Mechanopharmacology of Single Cells: A Step Towards Personalized Mechano-medicine

doi: 10.1002/adhm.201800069

Figure Lengend Snippet: Bronchodilators dose-dependently inhibit tension signal in both normal and asthmatic ASM cells. A–B) Representative RICM and tension images of asthmatic ASM cells seeded on the RGD-Cy3-I27 tension sensor and treated with 100 μM of albuterol and 100 μM of isoproterenol for 15 min. Scale bar, 10 μm. C) Plots show the normalized tension signal, generated by individual cells before and after bronchodilator treatment. Line represents mean±SEM. from n = 17 cells for albuterol treatment, and n = 13 cells for isoproterenol treatment from four independent surface preparations ***P<0.001 and ****P<0.0001 by Wilcoxon signed-rank test. D) Representative RICM, integrin tension, and overlay of RICM and tension images for a single, asthmatic ASM cell adhered on the RGD-Cy3-I27 tension surface and treated with a stepwise addition of albuterol (0.01 nM–1 mM). Scale bar, 10 μm. E–F) Representative dose-dependent curves for (E) normal donors and (F) asthmatic donors with and without the presence of nicotine (50 μg/ml). R2 values associated with both fits were >0.98. EC50 was determined by sigmoidal fitting. Error bars represent SEM of EC50 values obtained from n = 10 cells for each donor collected from ten surfaces. G) Scatter plot quantifying EC50 values from dose-dependent curves of normal (n = 3 donors) and asthmatic ASM cells (n = 4 donors) stimulated with nicotine (50 μg/ml) for 72 h and doxycycline (4 μg/ml) for 24 h, cultured on the tension sensor and treated with the stepwise addition of the albuterol. Each circle represents data collected from a single cell. The red line indicates the mean, while the box shows the SEM, n.s. = not signifcant, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 using Student’s t-test. H) EC50 values were pooled from all normal and asthmatic donors with and without the addition of nicotine for 72 h, ****P<0.0001 by Mann Whitney test.

Article Snippet: ASM cells were transiently transfected with either GFP-actin, GFP-vinculin or GFP-paxillin using Lipofectamine® LTX with Plus ™ reagent by mixing 1 μg of DNA with Lipofectamine® LTX and Plus ™ Reagent for each well in a 24-well plate and incubated for 24–48 h. Western blot After overnight serum starvation, human ASM cells (Lonza, Switzerland) ± nicotine (50 μg/ml, 72 h) and human ASM cells isolated from healthy and asthmatic donors were cultured in serum-free media.

Techniques: Generated, Cell Culture, MANN-WHITNEY