Tnf β

Monoclonal antibodies to p65, and phospho-specific p65-NF-κB, MMP-9, CXCR4, cleaved-Caspase-3, and β1-Integrin, were obtained from R&D Systems (Heidelberg, Germany). Antibodies to β-actin were from Sigma-Aldrich (Taufkirchen, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence and anti-Ki67 were from Dianova (Hamburg, Germany), and alkaline phosphatase-linked antibodies for Western blotting were from EMD Millipore (Schwalbach, Germany). Monoclonal anti-ALDH1 was obtained from Acris Antibodies GmbH (Herold, Germany). Monoclonal anti-CD133 and anti-CD44 were purchased from Abcam PLC (Cambridge, UK). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), 4′,6-diamidino-2-phenylindole (DAPI), and alginate were from Sigma-Aldrich (Taufkirchen, Germany). TNF-β was purchased from eBiosciences (Frankfurt, Germany). Furthermore, TNF-β was given as a kind gift by Genetech (South San Francisco, CA, USA). Calebin A was a generous gift from Sabinsa Corporation (East Windsor, NJ, USA). Calebin A was diluted as 10,000 µM stock solution in dimethyl sulfoxide (DMSO) and further diluted in cell culture medium for experimental investigations. The final concentration of DMSO did not exceed 0.1% during the experiments.

Monoclonal antibodies to p65, as well as phospho-specific p65-NF-κB, MMP-9, CXCR4, cleaved-Caspase-3, β1-Integrin and PARP, were obtained from R&D Systems (Heidelberg, Germany). Antibodies to β-Actin were from Sigma-Aldrich (Taufkirchen, Germany). Secondary rhodamine-coupled antibodies for immunofluorescence and anti-Ki67 were from Dianova (Hamburg, Germany) and alkaline phosphatase-linked antibodies for Western blotting from EMD Millipore (Schwalbach, Germany). MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DAPI, 5-Fluorouracil (5-FU), alginate, BMS-345541 and dithiothreitol (DTT) were from Sigma-Aldrich (Taufkirchen, Germany). Stock solutions of BMS-345541 (1000 µM) and of DTT (1000 mM) were prepared in PBS and further diluted in normal cell culture growth medium to obtain final concentrations. Then, 5-FU was diluted as a 1000 µM stock solution in ethanol and further diluted in the cell culture medium. Final concentrations of ethanol did not exceed 0.1% during treatment. TNF-β was purchased from eBiosciences (Frankfurt, Germany). Further, TNF-β was given as a kind gift by Genetech (South San Francisco, CA, USA). Calebin A (CA), with a purity of 99.65%, was a generous gift from Sabinsa Corporation (East Windsor, NJ, USA). Epon for electron microscopy was purchased from Plano (Marburg, Germany).

Calebin A was given as a kind gift by Sabinsa Corporation, East Windsor, NJ, USA. Alginate, Curcumin, BMS-345541, dithiothreitol (DTT), anti-β1-Integrin and anti-β-actin were purchased from Sigma (Munich, Germany), Epon was from Plano (Marburg, Germany). Stock solutions of DTT and BMS-345541 were prepared in PBS and further diluted in normal cell culture growth medium to obtain final concentrations. Calebin A and Curcumin were prepared in dimethylsulfoxide (DMSO) as a 5 mM stock solution and stored in small aliquots at −20 °C. During treatment, concentrations of DMSO did not exceed 0.1%. TNF-β was purchased from eBiosciences (Frankfurt, Germany) and additionally TNF-β (specific activity of 50 million U/mg) was given as a kind gift by Genetech (South San Francisco, CA, USA). Antibodies to p65-NF-κB, CXCR4, Caspase-3 and MMP-9 were from R&D Systems (Heidelberg, Germany). Ki-67 and secondary antibodies for fluorescence labelling were from Dianova (Hamburg, Germany).