## 3 Results
### Pre-Treatment with PACAP6-38 Blocks Doxycycline- and Minocycline-Induced tPA Activity  
To assess if doxycycline- and minocycline-induced tPA and uPA activities were related to PAC1 receptor activation, RT4 cells pre-treated (or not) with PACAP6-38 (a PAC1 receptor antagonist, 10 \(\upmu\)M) were supplemented with PACAP (100 nM), doxycycline (100 ng/mL) or minocycline (100 ng/mL) for 24 h and enzymatic activities were assessed using zymography. Figure 3: Zymographic assessment of tPA and uPA activity of RT4 cells in response to PACAP, doxycycline or minocycline. (**A**) Representative zymogram and densitometric analyses showing the relative levels of (**B**) tPA and (**B**) after treatment with PACAP, doxycycline or minocycline for 24 h. Values reported (arbitrary units) are the mean \(\pm\) S.E.M. of two separate experiments, each run using distinct batches of cells (n = 4/group), measured using ImageJ software. *\(p\) < 0.05, **\(p\) < 0.01 or ***\(p\) < 0.001, as determined by ANOVA followed by [PERSON]’s _post-hoc_ test. As shown, lytic areas on gels showed a significant increase in tPA and uPA activities after treatment with PACAP (** \(p<0.01\) vs. Control for both tPA and uPA), doxycycline (* \(p<0.05\) for both tPA and uPA) and minocycline (** \(p<0.01\) for tPA and * \(p<0.05\) for uPA) (Figure 5A,B,B\({}^{\prime}\)). Pre-treatment with PACAP6-38 completely prevented PACAP-induced tPA or uPA activities (SS \(p<0.01\) vs. PACAP-treated cells). In cells supplemented with doxycycline or minocycline, PACAP6-38 pre-treatment significantly reduced tPA (SS \(p<0.05\) vs. doxycycline or minocycline treated cells, respectively) and uPA (SS \(p<0.05\) vs. doxycycline-treated and SS \(p<0.01\) vs. minocycline treated cells) (Figure 5A,B,B\({}^{\prime}\)). Pre-Treatment with PACAP6-38 Blocks Doxycycline- and Minocycline-Induced tPA and uPA Protein Expression Levels  
To appraise if pre-treatment with the PAC1 receptor antagonist PACAP6-38 prevented doxycycline and/or minocycline-induced expression of plasminogen activators, we performed Western blot analyses in RT4 cells that were treated as per zymographic analyses. A statistical significant increase in tPA protein expression was observed both in PACAP- (* \(p<0.05\) vs. Ctrl), doxycycline- or minocycline-treated cells (* \(p<0.05\) for both, Figure 6A,A\({}^{\prime}\)). Figure 4: Immunocytochemistry of tPA in RT4 cells exposed to PACAP, doxycycline or minocycline. Representative cytochemistry images showing tPA immunoreactivity in (**A**) negative control (no primary Ab), (**B**) untreated, (**C**) PACAP treated, (**D**) doxycycline treated and (**E**) minocycline treated RT4 cells. Values are reported as percentage of tPA-positive cell surface area normalized to total cell surface area \(\pm\) S.E.M. **\(p<0.01\) or *** \(p<0.001\), as determined by ANOVA followed by [PERSON]’s _post-hoc_ test. Magnification \(=20\times\). Scale bar \(=60\) μm. Figure 5: Zymographic assessment of tPA and uPA activity of RT4 cells in response to PACAP, doxycycline or minocycline with and without PACAP6-38 pre-treatment. (**A**) Representative zymogram and densitometric analyses showing the relative levels of tPA (**B**) and uPA (**B’**). Cells were treated with either PACAP, doxycycline or minocycline only or pre-treated for 1 h with PACAP6-38 and then assessed after 24 h. Values reported (arbitrary units) are the mean \(\pm\) S.E.M. of two separate experiments, each run using four separate batches of cells (n = 4 / group), measured using ImageJ software. * \(p\) < 0.05 or ** \(p\) < 0.01 vs. Control; $ \(p\) < 0.05 or $S \(p\) < 0.01 vs. corresponding drug-treatment groups, as determined by ANOVA followed by Sidak’s _post-hoc_ test. Pre-treatment with PACAP6-38 prevented the induction of tPA expression by PACAP (SS \(p<0.01\) vs. PACAP-treated), doxycycline (SSS \(p<0.001\) vs. doxycycline-treated) and minocycline (SS \(p<0.01\) vs. minocycline-treated). Similar effects were seen when analyzing uPA protein expression.