Ki 67

Tissue sections of 2 µm from representative paraffin blocks were stained. Briefly, for antigen retrieval, the sections were heated in citrate buffer at pH 6 in a steamer for 20 minutes. The primary antibody was used in the below-cited concentrations in phosphate buffer saline (PBS); slides were incubated with 50 µl per section in a humid chamber at room temperature for 30 minutes. As a detection system, we used the EnVision Kit (Dako, Carpinteria, CA, USA) according to the manufacturer's protocols. The proportion of cells showing a positive staining was categorized as follows: “no staining detected” (−); “staining in up to 30%” (+); “staining in more than 30% and up to 70%” (++) and “staining in more than 70%” (+++) of the total number of tumour cells analysed.
The following antibodies were used: CDK4 (1 : 100, Zytomed Systems), CDK6 (1 : 100, Abcam), Phospho-Rb (Ser780; 1:250, Cell Signaling Technology), p16 (1 : 100, Santa Cruz Biotechnology), Ki-67 (1 : 200, clone MIB-1, Dianova, Hamburg, Germany), and cleaved caspase-3 (Asp175, 1 : 1005A1E, Cell Signaling Technology, 9664).

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).