This extra density is also visible in the activated form of the molecule, albeit to a lesser extent

ibody anti-Bax, anti-Bcl-2, anti-procaspase, anti-caspase-3 for apoptosis studies and anti-connexin-43, anti-phospho-connexin-43, antiFocal Adhesion Kinase and anti-phosho-Focal Adhesion Kinase for cell migration studies; a-tubulin was used as reference protein. After incubation with the primary antibodies and washing in TBS/0.1% Tween, the appropriate secondary antibody, either anti-mouse or anti-rabbit, was added for 1 h at room temperature. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemiluminescence reagents and exposed to Hyperfilm. Films were then subjected to densitometric analysis using a Gel-Doc 2000 system. order Vesnarinone results NIV and DON Affect IEC-6 Viability After incubation of IEC-6 cells with graded concentrations of NIV or DON alone or in combination for 24 h, cell viability was determined using the MTT assay. Control cells viability was designated as 100% and results have been expressed as the concentration of each mycotoxin which induces 50% of mortality in IEC-6. Both mycotoxins exerted a significant effect on cell viability and had anti-proliferative activity on IEC-6. In particular NIV exerted a stronger anti-proliferative activity compared to DON. Moreover, the presence of NIV and DON together in the incubation medium did not induce either additive or synergistic effects on IEC-6 viability. NIV- and DON-induced Apoptosis in IEC-6 Cells In order to investigate the mechanism underlying cell observed decrease in cell viability observed in NIV- or DONtreated IEC-6 cells a cytofluorimetric analysis was performed by incubating IEC-6 with graded concentrations of each mycotoxin for 24h. Apoptosis was evaluated by cytofluorimetric analysis of PI stained hypodiploid nuclei. Our results indicate that both NIV and DON significantly induced apoptosis in IEC-6, in a concentration-dependent manner, from 5 mM and 10 mM for NIV- or DON-treated cells respectively; whereas any apoptotic effect have been observed at the lowest concentrations tested. Interestingly the NIV pro-apoptotic effect at concentrations of 10 80 mM was significantly higher than DON’s, indicating a stronger pro-apoptotic activity for NIV. Similarly to the effect observed on cell viability, no additive or synergistic effects on IEC-6 apoptotis were observed after incubation with NIV and DON together. Wound-healing Assay IEC-6 cells were seeded in a 12-well plastic plate at 1.86105 cells per well and allowed to adhere; after 24 h of serum deprivation a wound was produced 19778726 at the centre of the monolayer by gently scraping cells with a sterile plastic p200 pipette tip. Then the incubation medium was removed and cells washed with PBS and incubated with NIV and DON, alone or in combination, at non apoptotic concentrations. The wounded cell cultures were then incubated at 37uC in a humidified and equilibrated incubation chamber of an Integrated Live Cell Workstation Leica AF-6000 LX. A 106 phase contrast objective was used 19535597 to record cell movements with Induction of Nuclear Morphological Changes in IEC-6 by NIV and DON In order to confirm induction of apoptosis and perturbation in cell morphology due to the trichothecenes NIV or DON, IEC-6 nuclei were also observed after DAPI staining. At the lowest concentrations tested cell nuclei appeared similar to untreated cells. Apoptotic cells were clearly identified by the appearance of pyknotic nuclei and apoptotic bodies after incubation with the highest NIV and DON Affect IEC-6 Homeostasis NIV concentr