Caspase 3

In preparation for examination of fixed tissues, tumors were preserved in 10% neutral buffered formalin. Fixed tissues were processed for paraffin embedding, sectioned and stained. Immunohistochemical staining was performed with antibodies to human p53 (Leica Biosystems, Newcastle Upon Tyne, UK; Novocastra mouse monoclonal DO-7), Mdmx (Bethyl Laboratories, Inc.; A300 287A), Mdm2 (Thermo Scientific, Neo Markers, Fremont, CA, USA), caspase-3 (R&D Systems, Minneapolis, MN, USA; Monoclonal Mouse 84803) and ki67 (Cell Marque, Sigma-Aldrich company, Carlsbad, CA, USA; rabbit monoclonal, SP6). Histopathological analyses were performed by an onco-pathologist (JMP). Photographs were taken using a using a BX-50 microscope with Leica DFC290HD camera (Olympus, Notting Hill, VIC, Australia) and Leica Application Suite V3.8.0 software. IHC scoring was undertaken using BX-41 microscope (Olympus).
Fresh-frozen tissues were prepared from OCT-embedded tissues snap frozen in isopentane liquid nitrogen vapors and cryostat sections were stained for β-gal (essentially as in Dimri et al.^{46 (link)}). Images were visualized using a BX- 51 microscope (Olympus). β-Gal staining of cultured cells was performed as published.^{46 (link)} Images were visualized using a Zeiss Axio Vert.A1 microscope (Zeiss, Peabody, MA, USA) and acquired using SPOT Version 4.7 software (Diagnostic Instruments).

As part of our experiments, the following antibodies were used: Beclin-1 (#3738) and LC3-II (#4108) from Cell Signaling Technology (Danvers, MA, USA); NF-κB (#MAB5078), MMP-9 (#MAB911), and caspase-3 (#AF835) from R&D Systems (Heidelberg, Germany); Sox9 (#TA802387) from OriGene Technologies (Herford, Germany); β-actin (#A4700), collagen type II (#AB761), and CSPG (#MAB5384-I) from Sigma-Aldrich (Taufkirchen, Germany); PI3K (#ab154598), Akt (#ab38449), and mTOR (#ab109268) from Abcam (Berlin, Germany); secondary immunofluorescence antibodies from Dianova (Hamburg, Germany); and secondary Western blot antibodies from EMD Millipore (Schwalbach, Germany). Furthermore, TNF-α was from R&D Systems (Heidelberg, Germany), 3-methyladenine (3-MA) from VWR International (Ismaning, Germany), and Epon from Plano (Marburg, Germany). BMS-345541, Fluoromount, DAPI, and MTT reagent were from Sigma-Aldrich (Taufkirchen, Germany). CA from Sabinsa Corporation (East Windsor, NJ, USA) was prepared as 5,000 µM stock in dimethyl-sulfoxide (DMSO) solution. The experimental concentrations were further diluted in cell culture medium without exceeding a DMSO concentration of 0.1%.

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).

Serum ALT, AST and LDH were measured in the Institute of Clinical and Laboratory Medicine at the University Hospital Heidelberg according to standard procedures.
For detection of caspase activity, caspase-3, caspase-8 and caspase-9 fluorometric assays (R&D Systems, Wiesbaden, Germany) of protein extracts of homogenated tissue from post ischemic livers were performed, as previously described^{25 (link)}.

Ac-K(Alloc)GD(All)E(All)VD(All)-OH (alloc-KGDEVD) was purchased from Peptron (Daejeon, Korea). Chlorin e6 (Ce6) was purchased from Frontier Scientific Inc. (Logan, UT, USA). 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ), Bis(p-nitrophenyl) carbonate, 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), Dimethyl sulfoxide (DMSO), N,N-diisopropylethylamine (DIPEA), hydroxybenzotriazole (HOBt), p-aminobenzyl alcohol, tetrakis(triphenylphosphine)palladium, and N-hydroxysuccinimide (NHS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anhydrous dimethylformamide (DMF) and dimethylsulfoxide (DMSO) were purchased from Merck (Darmstadt, Germany). Tributyltin hydride (Bu3SnH) and glacial acetic acid were purchased from Acros (St. Louis, MO, USA). DMEM medium, penicillin–streptomycin, and fetal bovine serum (FBS) were purchased from WelGENE Inc. (Daegu, Korea). Cathepsin B, D, K, L, caspase-9, caspase-3, and caspase-3 ELISA Kit (cat# SMIF00) were purchased from R&D system (Minneapolis, MN, USA). KPC960 (Human pancreatic cancer) cell lines were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). Tem grid (Carbon Film 200 Mesh copper) was purchased from Electron Microscopy Sciences (Hatfield, PA, USA). Cell counting kit-8 (CCK-8) was purchased from Vitascientific (Beltsville, MD, USA).