TRPV1 was involved in inhibition of secretion and intracellular accumulation of MMP-9 upon WIN-treatment.

<p>(a,b) Western blot analyses of U937-macrophage cell lysates (MMP-9 cellular) using MMP-9 antibody. (a) Treatment with the TRPV1 antagonist capsazepine (CZP) enhanced the WIN-induced size shift of MMP-9 from 85 to 92 kDa when given parallel to WIN and mimicked this effect when administered separately. The figure shows one representative analysis out of three. (b) Treatment with the TRPV1 agonist capsaicin (CIC) antagonized the WIN-induced size shift while it exhibited no effect when given alone. The figure shows one representative analysis out of three. (c) MMP-9 activity–ELISA of conditioned medium. The WIN-induced decrease of MMP-9 activity was intensified by CZP (10 µM), and antagonized by CIC (10µM). Data are shown as means +/− SD, n = 3. **p<0.001 *p<0.1 according to Newman-Keuls Multiple Comparison test following ANOVA.</p

DNA integrity after transformation.

<p>Recovered plasmid DNA was transformed in <i>E coli</i> KRX and the integrity was calculated based on the transformation efficiency in colonies per nanogramm DNA. A significant increase in the degree of DNA integrity was detected for the samples applied on the bottom side of the payload compared to the surface samples (p-value = 0.01). (surface samples: n = 4; screw heads samples: n = 8; bottom side samples: n = 3; positive control: n = 1, original sample: n = 1) (SD, two-tailed unpaired t-test; p-values are shown for respective pairwise analyses).</p

Flight induced mutagenesis anlysis by sequencing.

<p>Flight induced mutagenesis anlysis by sequencing.</p

TEXUS-49 telemetry flight data.

<p><b>a</b> altitude, <b>b</b> acceleration, <b>c</b> temperature and <b>d</b> pitch, roll and yaw angles of rotation.</p

Glycosylation of intracellular 92 kDa-MMP-9 after WIN-treatment was different from the 85 kDa-MMP-9 in untreated U937-macrophages.

<p>The pictures show one representative analysis out of three. (a) Western blot analysis using anti-MMP-9 antibody of endoglycosidase H-digested cell lysates treated with or without WIN. The WIN-induced 92 kDa-MMP-9 was resistant to digestion, whereas the 85 kDa-MMP-9 from control cells loses 5 kDa upon digestion. (b) Digestion with N-glycosidase F resulted in a loss of 5 kDa in both MMP-9 forms.</p

Treatment with WIN reduced MMP-9 protein in bronchoalveolar lavage fluid (BALF) of mice with smoke-induced lung inflammation.

<p>Mice were exposed to air, cigarette smoke (smoke), or cigarette smoke plus i.p. treatment with 5 mg/kg/d WIN (smoke + WIN). (a) MMP-9 protein was measured by ELISA in BALF. Cigarette smoke-exposure enhances the MMP-9-content of BALF. I.p application of WIN during cigarette smoke-exposure reduced MMP-9 in BALF. (b) Number of white blood cells (WBCs) in BALF measured by haemocytometry. Cigarette smoke-exposure enhanced the content of WBCs in BALF significantly. I.p. application of WIN during cigarette smoke-exposure did not alter the number of WBCs. (c) Ratio of MMP-9/10⁵WBCs. The amount of MMP-9 per WBC decreased upon i.p. application of WIN significantly. Data are shown as means +/− SD, n = 7 (air) n = 8, (smoke), n = 9 (smoke+WIN). *p<0.05, ^#p>0.05 according to Newman-Keuls Multiple Comparison test following ANOVA.</p

DNA recovery rate after re-entry and landing.

<p>After post-flight payload retrieval, DNA samples were recovered and dissolved in Tris buffer. The DNA concentration was measured and the recovery rate was calculated with respect to the initial application concentration. DNA was detected in all the analyzed samples pc = positive control; nc = negative control <b>a</b> DNA recovery rate at single sample positions, <b>b</b> average DNA recovery rate for surface (n = 4), screw head (n = 8) and bottom side (n = 3) (SD, two-tailed unpaired t-test; p-values are shown for respective pairwise analyses).</p

WIN-induced regulation of MMP-9 was mediated by a specific binding site, which is different from CB1, CB2, and PPARy, and independent from pertussis-toxin.

<p>Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) of U937-macrophages treated with the receptor-inactive WIN-enantiomer S(–)-[2,3-Dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1 naphthalenyl) methanone mesylat (WIN3), specific inhibitors for cannabinoid-receptors or pertussis toxin (PTX). Control cells were treated with vehicle. In each case the figure shows one representative analysis out of three. (a) Treatment with WIN3 demonstrated the specificity of the effect of WIN. (b) Inhibitors for CB1 (AM251) and CB2 (AM630) did not abolish the WIN-induced inhibition of secretion and intracellular accumulation of MMP-9. (c) Treatment with PTX did not abolish the WIN-induced effect. (d) Inhibition of PPARy with GW9662 had no influence on the effect of WIN on MMP-9.</p

Inhibition of MMP-9 secretion was accompanied by an intracellular accumulation of a 92 kDa-MMP-9.

<p>Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted) of U937-macrophages treated with WIN using anti-MMP-9 antibody. Control cells were treated with vehicle. (a) Upon 24 h treatment with WIN, the amount of MMP-9 in the cell lysate increased, whereas the amount of secreted MMP-9 in the conditioned medium decreased. The size of MMP-9 in the cell lysate shifted from 85 kDa to 92 kDa. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) Kinetic analysis of MMP-9 after WIN-treatment throughout 24 h. The inhibition of MMP-9 secretion was accompanied by an accumulation of intracellular 92 kDa-MMP-9. The intracellular 85 kDa-MMP-9 disappeared with time. The figure shows one representative analysis out of three.</p