Time course of transcription factor induction after stimulation with TPA and ionomycin

<p><b>Copyright information:</b></p><p>Taken from "Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors"</p><p>http://www.biomedcentral.com/1471-2172/8/18</p><p>BMC Immunology 2007;8():18-18.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018703.</p><p></p> ABL and CBL were harvested at different time points by lysing cells. mRNA levels were determined for T-bet (A), GATA-3 (B), and c-maf (C). Data points are mean ± standard error for 6 donors. *< 0.05 ABL vs. CBL

Kinetics of cytokine induction after stimulation with TPA and ionomycin

<p><b>Copyright information:</b></p><p>Taken from "Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors"</p><p>http://www.biomedcentral.com/1471-2172/8/18</p><p>BMC Immunology 2007;8():18-18.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018703.</p><p></p> ABL and CBL were harvested at different time points by lysing cells. mRNA levels for IFNγ (A), IL-4 (B), and IL-3 (C) were determined as in Figure 1. Data points are the mean ± standard error for 6 donors. *< 0.05, **< 0.01 ABL vs. CBL

Induction of transcription factors in ABL and CBL after stimulation for 4 hrs

<p><b>Copyright information:</b></p><p>Taken from "Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors"</p><p>http://www.biomedcentral.com/1471-2172/8/18</p><p>BMC Immunology 2007;8():18-18.</p><p>Published online 31 Aug 2007</p><p>PMCID:PMC2018703.</p><p></p> mRNA levels of T-bet (A), GATA-3 (B), and c-maf (C) were determined using real-time RT-PCR. Data are expressed as arbitrary units normalized to β-actin to correct for RNA quantity and integrity. Columns are the mean ± standard for 3 donors. The inset shows the correlation between expression of the respective transcription factor with IFNγ and IL-4. Dotted line indicates basal transcript levels. Iono – Ionomycin

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

Treatment with WIN reduced secretion and activity of MMP-9 in macrophageal differentiated U937 cells.

<p>(a) Western blot analysis of conditioned medium using antibodies against MMP-9 and MMP-12. WIN-treatment resulted in a significant decrease of secreted MMP-9, whereas MMP-12 secretion was not affected. Control cells were treated with vehicle. The figure shows one representative analysis out of three. (b) MMP-9 activity-ELISA of conditioned medium. Upon treatment with 2 µM WIN a reduction of MMP-9 activity was observed, after treatment with 4 µM WIN the reduction was even stronger. Control cells were treated with vehicle. Data are shown as means +/− SD, n = 3. *p<0.05 vs. control, **p<0.01 according to Newman-Keuls Multiple Comparison test following ANOVA. (c) Zymography of conditioned medium. Gelatinolytic activity was strongly decreased by 2 and 4 µM WIN. The figure shows one representative analysis out of three.</p

Inhibition of MMP-9 secretion and activity and intracellular accumulation of MMP-9 in WIN-treated osteoclasts, but not in microglia.

<p>(a) Western blot analysis of cell lysates (MMP-9 cellular) and conditioned medium (MMP-9 secreted). using anti-MMP-9-antibody and MMP-9-activity ELISA of conditioned medium (bar chart) from osteoclasts. Upon WIN treatment (4 µM), the amount of intracellular 92 kDa-MMP-9 was enhanced, while the amount of secreted MMP-9 and the activity of MMP-9 in the conditioned medium was decreased. (b) Western blot analysis of cell lysates (MMP-9 cellular) using anti-MMP-9-antibody and MMP-9 ELISA of conditioned medium (bar chart) from primary microglia. Size and amount of intracellular MMP-9 were not changed after WIN-treatment (4 µM). The amount of MMP-9 in the conditioned medium increased insignificantly. PC = positive control (U937 macrophages). The figure shows one representative analysis out of three. Data are shown as means +/− SD n = 3. *p<0.05, ^#p>0.05 vs. control according to unpaired t test.</p

WIN reduced bone resorption and MMP-9-activity in a capsaicin sensitive manner.

<p>(a) Measurement of resorption activity of osteoclasts using crosslaps-ELISA of conditioned medium. Treatment with WIN (4 µM) reduced the osteolytic activity compared to control cells (vehicle treated). Additional treatment with capsaicin (CIC) antagonized this decrease. Data are shown as means +/− SD, n = 5. (b) MMP-9-activity-ELISA of conditioned medium of osteoclasts. Treatment with WIN (4 µM) decreased MMP-9-activity significantly compared to control cells (vehicle treated) and this decrease was antagonized by parallel treatment with CIC. Data are shown as mean +/− SD, n = 5. *p<0.05, ^#p>0.05 according to Newman-Keuls Multiple Comparison Test following ANOVA.</p

Dephosphorylation of ERK1/2 is involved in the WIN-induced down regulation of MMP-9 mRNA, but not in the effect of WIN on MMP-9 protein.

<p>MMP-9 quantitative Real-time PCR (bar chart) and Western blot analyses of intracellular (MMP-9 cellular) and secreted (MMP-9 secreted) MMP-9 and for phosphorylated (pERK1/2) and unphosphorylated ERK1/2. The figure shows one representative analysis out of three. U937-macrophages were treated with WIN (4 µM) or the pharmacological ERK1/2 phosphorylation inhibitor U-0126 (50 µM). Lane1: control; lane 2: WIN-treatment; Lane 3: U0126-treatment; lane 4: WIN + U0126-treatment. WIN treatment resulted in a dephosphorylation of ERK associated with inhibition of secretion, intracellular accumulation and decrease of MMP-9 mRNA (lanes 1 and 2). Inhibition of ERK1/2 phosphorylation with U-0126 decreased the level of MMP-9 mRNA significantly, but did not affect MMP-9 protein (lane 3). Treatment with WIN and U-0126 together did not decrease MMP-9 mRNA further than caused by U-0126 alone (lines 3 and 4). Bar chart: Data are shown as means +/− SD n = 3. *p<0.01 vs. control, ^#p>0.05 according to Newman-Keuls Multiple Comparison test following ANOVA. Western blot: ß-actin 1 is from the same blot as p-EKR1/2, ß-actin 2 is from the same blot as MMP-9 and ERK1/2. Originally, the blots contained more samples. In order to arrange the figure for comparison with mRNA quantification, blots were cut and rearranged. Original lanes can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048272#pone.0048272.s001" target="_blank">Figure S1</a>.</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