Gels were digitized using GeneTools software. Immunohistochemistry hCMECD3 cells grown on glass coverslips were fixed in 4% PFA and were incubated for 1 h at room temperature ARQ197 FDA with rabbit anti ZO 1 or goat anti MMP 9 primary poly clonal antibodies. Subsequently, cells were incubated with Alexa Fluor 488 or 594 anti mouse or anti goat secondary antibodies followed by Hoechst and mounted in fluorescent mounting medium. The mounted Inhibitors,Modulators,Libraries slides were observed with a Leica TCS SP2 confocal microscope. High magnification images were acquired using a 633 HCX PL APO oil immersion objective by sequential scanning to minimize the crosstalk of fluorophores. For each channel, photo multiplier gains and offsets were adjusted to use full image dynamic range.

Inhibitors,Modulators,Libraries Images acquired at 1,024 1,024 pixels and saved in TIFF format were processed for colo calization analysis using ImageJ plug in processing soft ware. Image editing was performed using Adobe Photoshop. MAPK inhibition studies To investigate the involvement of mitogen activated pro tein kinases in MMP 9 expression following TWEAK stimulation, we blocked c RAF1 and MEK sig naling pathways using ERK2 and MEK12 specific inhi bitors at working concentrations of 5 uM GW5074 and 0. 5 uM U0126v. hCMECD3 cells were cultured for 24h in the presence or absence of TWEAK and in the presence or absence of the MAPK inhibitors cell lysates and supernatants were then collected and tested by gel zymography. Results hCMEC express Fn14 and are a target of TWEAK In a first step, we used RT PCR to study, in the hCMECD3 cells, the expression of the mRNAs encoding TWEAK and its receptor Fn14.

We found Inhibitors,Modulators,Libraries that hCMECD3 cells express both TWEAK and Fn14 mRNAs. We next used flow cytometry to assess TWEAK and Fn14 expression at the membrane in the same cells. We show that hCMECD3 cells do not consti tutively express membrane TWEAK but constitutively ex press Fn14 on their surface. TWEAK exposure did not up regulate TWEAK or Fn14 expression at the cell surface. Similarly, using ELISA, we were not able to detect Inhibitors,Modulators,Libraries soluble TWEAK in the culture supernatants of hCMECD3. In agreement with data issued from Western blot analysis of TWEAK expression by hCMECD3 cells, these results lead us to conclude that this cell line expresses neither membrane nor soluble TWEAK. TWEAK induces inflammation of HCMEC Next, we studied the effects of TWEAK on hCMECD3 proliferation using a BrdU incorporation test.

As indi cated in Figure 2, a 24h TWEAK exposure of the cells induced proliferation. It is worth noting that the prolif erative effects of soluble TWEAK are significantly higher than those of TNF. To assess the potential inflammatory effects of TWEAK on hCMECD3 cells and BBB inflammation, we used ELISA to measure cytokine secretion in the cul ture supernatants after TWEAK or TNF exposure Inhibitors,Modulators,Libraries dur ing 24h, compared with buy inhibitor nonstimulated cells.