hbsmc  (PromoCell)

Journal: PLOS One

Article Title: β-Nicotinamide adenine dinucleotide (β-NAD) acts as a bronchodilator

doi: 10.1371/journal.pone.0334491

Figure Lengend Snippet: (A, B) Force recordings in organ bath, β-NAD (A) and salbutamol (B) concentration-dependently relax precontracted human bronchioli. n refers to the number of bronchioli, followed by the number of lungs from which they were taken, presented as bronchioli/lungs. (C) Recording of [Ca 2+ ] i in HBSMC with Fura-2 AM. Both β-NAD and ATP cause rise in [Ca 2+ ] i . n refers to the number of cells, taken from 4 independent experiments.

Article Snippet: HBSMC (C-12561, PromoCell, Heidelberg, Germany) were cultured in Smooth Muscle Cell Growth Medium 2 for at least 24 h at 37°C.

Techniques: Concentration Assay

Journal: PLOS One

Article Title: β-Nicotinamide adenine dinucleotide (β-NAD) acts as a bronchodilator

doi: 10.1371/journal.pone.0334491

Figure Lengend Snippet: (A, B) Recording of intracellular cAMP concentration in HBSMC via FRET, with low FRET ratio indicating high cAMP concentration. β-NAD and isoproterenol cause a decrease in FRET ratio, reflecting rise in intracellular cAMP concentration. In the presence of KH7 (30 µM), a soluble adenylyl cyclase antagonist, the cAMP response to β-NAD was blocked, while the isoproterenol-induced cAMP increase remained unaffected. (A) FRET ratio over time and (B) ΔFRET ratio (%) represents the change in response to β-NAD and isoproterenol, measured in the presence and absence of KH-7. Within-group comparisons include the ΔFRET ratio before agonist addition versus after the addition of β-NAD or isoproterenol. n represents the number of cells analyzed, derived from three independent experiments. Error bars indicate mean ± SEM throughout. Statistical analysis was performed using the Wilcoxon signed-rank test (two-tailed) with Bonferroni correction for multiple comparisons. (C-J) Force recording from trachea in organ bath (C-F) and videomorphometric recording of luminal bronchial area in PCLS (G-J) in specimens taken from mice lacking the C1 (C, E, G, I) or C2 domain of soluble adenylyl cyclase (D, F, H, J). In both assays, β-NAD-induced relaxation of muscarine-precontracted airways was not significantly reduced in knockout mice compared to their respective wildtype controls. (G-J) In PCLS, KH7 (30 µM) has no significant effect upon muscarine-induced contraction and β-NAD-induced relaxation, both in knockout and in wild-type mice. (E, F, I, J) Scatterplots depict changes induced by β-NAD related to the preceding response to muscarine. (E, F) Scatterplots show the β-NAD-induced relaxation effect (%) relative to the muscarine response. (I, J) Scatterplot showing the maximum peak responses of the second stimulation (first response set as 100%) in the presence of KH7 within C1 and C2 knockout groups and their corresponding wild-type controls. The corresponding controls with the application of vehicle (DMSO) instead of KH7 are depicted in . Statistical analysis was performed using the Mann-Whitney test. Data are expressed as mean ± SEM. n refers to the number of animals.

Article Snippet: HBSMC (C-12561, PromoCell, Heidelberg, Germany) were cultured in Smooth Muscle Cell Growth Medium 2 for at least 24 h at 37°C.

Techniques: Concentration Assay, Derivative Assay, Two Tailed Test, Knock-Out, MANN-WHITNEY

Journal: Nature Communications

Article Title: Maternal and perinatal obesity induce bronchial obstruction and pulmonary hypertension via IL-6-FoxO1-axis in later life

doi: 10.1038/s41467-022-31655-z

Figure Lengend Snippet: For the experiments with STAT3-inhibitor (Stattic) and AKT inhibitor (AKT-Inh.), hbSMC were pre-exposed to either Stattic, AKT-Inh. or vehicle for 2 h. Subsequently, the hbSMC were stimulated as indicated. A Immunoblot showing phosphorylated STAT3 (pSTAT3) in hbSMC after exposure to IL-6 with soluble IL-6 receptor (IL-6 + R) or vehicle for 30 min. B Immunoblot showing pAKT and total AKT in hbSMC after exposure to vehicle, IL-6 + R, or Stattic for 30 min. β-actin was used as a loading control. A densitometric summary is displayed. C Nuclear and cytoplasmatic phosphorylated FoxO1 (pFoxO1) fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with IL-6 + R alone or combined with AKT-Inh. or vehicle for 30 min. D Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with insulin alone or combined with AKT-Inh. or Stattic. E Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per hbSMC using immunofluorescent staining; hbSMC were treated with IL-6 + R or insulin alone or both in combination with AKT-Inh. or Stattic for 30 min. F Viability (MTT assay) of hbSMC after exposure to vehicle, IL-6 + R, Paclitaxel and/or Stattic. G – I hbSMCs were exposed to IL-6 + R or vehicle with or without Paclitaxel for 30 min ( G ) or 24 h ( H , I ). Nuclear and cytoplasmatic pFoxO1 fraction in % of total cell pFoxO1 per bSMC ( G ); total FoxO1 per hbSMC ( H ), and proliferation (BrdU) of hbSMC ( I ). Data are shown as mean ± standard error of the mean. A – I n = 3–5 biological independent samples or experiments per group; (repeated measure) one-way ANOVA followed by the Bonferroni post-test or the two-sided Mann–Whitney test; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Gray = vehicle; white = stimulation; orange = Paclitaxel; blue = IL-6. A detailed list of sample sizes and P value per graph is provided in the Supplemental Material. Source data are provided as a Source Data file.

Article Snippet: Human bronchial SMC (hbSMC) (PromoCell; #397Z013.3, #C-12561, Heidelberg, Germany) and human aortal SMC (haSMC) (ATCC, #PCS-100-012, Manassas, VA, USA) were cultured according to the manufacturer’s recommendations (PromoCell; Smooth Muscle Cell Growth Medium 2: Lot#427M002; Supplement-Mix: Lot#425M235, Heidelberg, Germany).

Techniques: Western Blot, Control, Staining, MTT Assay, MANN-WHITNEY