M30 cytodeath

All serologic measurements were performed at Imperial College NHS Trust Clinical Laboratories. Serum insulin, CRP, and IGF‐1 were analyzed using enzyme‐linked immunosorbent assays. Markers of apoptosis (M30), colonocyte proliferation (Ki‐67), and insulin signaling pathway activation (phospho‐mTOR) were assessed using immunohistochemical staining in the laboratory of Professor Robert Goldin, Imperial College, and Dr Naomi Guppy, University College London. All staining was performed using the Leica Bond III automated immunostaining platform on the Leica Bond Polymer Refine (Leica, DS9800) DAB polymer detection system. Ki‐67 (mouse monoclonal MIB‐1, Dako, cat. no. M7240) was applied at a dilution of 1/120 for 15 minutes at room temperature, following on‐board heat‐induced epitope retrieval (HIER) using Leica Epitope Retrieval 2 (ER2, pH9 retrieval solution; Leica, AR9640) for 20 minutes. M30 (mouse monoclonal M30 cytoDEATH, Roche, cat. no. 12 140 322 001) was applied at a dilution of 1/50 for 30 minutes at room temperature, following on‐board HIER using Leica Epitope Retrieval 1 (ER1, pH6 retrieval solution; Leica, AR9961) for 30 minutes. Phospho‐mTOR (rabbit monoclonal 49F9, Cell Signaling Technologies cat. no. 2976) was applied at a dilution of 1/50 for 20 minutes at room temperature, following on‐board HIER using Leica ER2 for 30 minutes.

Native rat PFN was purified from RNK16 cells and native hGzmB was purified from YT-Indy cells as described³⁴ . GzmA was purified as described³⁵ . MDA-MB231 cells ± CP-31398 were resuspended in HBSS (Gibco) containing 10 mM Hepes pH 7.5, 4 mM CaCl₂, 0.4% BSA before adding sublytic dose of PFN ± 100 nM hGzmB or 500 nM GzmA, diluted in HBSS containing 10 mM Hepes pH7.5. To assess PFN/GzmB-induced apoptosis, cells incubated for 2 h at 37 °C with buffer or sublytic PFN ± 100 nM hGzmB were analyzed for caspase activation by flow cytometry using M30-FITC mAb staining according to the manufacturer protocol (M30 CytoDEATH, Roche) to detect an effector caspase-cleavage product of cytokeratin-18^{36 (link)}. To assess PFN/GzmA-induced cell death, cell were analyzed by flow cytometry after Annexin-V/PI staining, as described above.

As a more specific method, Caspase assay was used to confirm the results. Hereby apoptosis was evaluated by measuring the level of caspase-cleaved cytokeratin 18 (M30, Roche, Switzerland; order number: 121140322001). The ovarian cell lines A2780, cisA2780 and UWB1.289 were seeded at a density of 1.0 × 10⁴ cells/well on 96-well plates in 200 μl medium. After 20 h, 50 or 100 µM RSV were added and cells were incubated for 24 h. M30 CytoDeath (Roche, Switzerland; order number: 121140322001, dilution 1:1000) was used to detect the apoptotic cells.

To assess PFN/GzmB-dependent apoptosis, T1 cells incubated for 2 hr at 37°C with buffer or sublytic PFN ± 50 nM hGzmB, were analyzed for caspase activation by flow cytometry using M30-FITC mAb staining (M30 CytoDEATH, Roche) to detect an effector caspase-cleavage product of cytokeratin 18, as previously described [37 (link), 38 (link)]. Native rat PFN was purified from RNK16 cells and native hGzmB was purified from YT-Indy cells as described [64 ].

The IHC staining was performed using a Histofine Kit (Nichirei, Tokyo). The positivity of ER and progesterone receptor (PgR) was defined by ≥10% nuclear staining. The expression of human epidermal growth factor receptor 2 (HER2) was examined using the HercepTest (Dako, Tokyo). HER2-positivity was defined as either 3+ or 2+ with HER2 gene amplification by fluorescence in situ hybridization. The autophagy- and apoptosis-related markers were stained using the anti-beclin 1 antibody (1:250; NB500-249; Novus Biologicals, CO, USA), anti-LC3 antibody (1:200; PM036; MBL, Nagoya, Japan), TUNEL (In Situ Cell Death Detection Kit; Roche Diagnostics, Mannheim, Germany) and M30 CytoDEATH (1:100; Roche Diagnostics). The cytoplasmic staining of beclin 1, LC3, and M30 and nuclear staining of TUNEL were assessed with pre- and post-treatment tissues. The expression rate of each marker was assessed as positive cancer cells per total cancer cells.

Protein extracts were separated on SDS/PAA gels, transferred onto Amersham Hybond-P PVDF membranes and incubated with primary antibodies as mentioned19 (link). The following antibodies were used: Wnt5a (C27E8, Cell Signaling, 1:1000), GAPDH (14C10, Cell Signaling 1:5000), α-tubulin (Calbiochem DM1A, 1:5000), actin (Sigma 1:2500) CCNA (clone 6E6, Thermo Scientific, 1:100), CCD1 (DSC-6 Santa Cruz 1:500) CD14 (Atlas Antibodies, HPA001887, 1:200) CD56 (123C3, Cell Signaling 1:1000), phospho-p44/p42 MAPK (ERK1/2) (Thr202/Tyr204) (D13.14.4E XP, Cell Signaling, 1:1000), p44/42 MAPK (ERK1/2) (Cell Signaling, 1:1000), phospho-PKC (pan) (βII Ser660) (Cell Signaling, 1:1000), phospho-AKT (Ser473) (D9E XP, Cell Signaling 1:1000), caspase-cleaved cytokeratin 18 neo-epitope (M30 CytoDEATH, Roche, 1:500), cleaved caspase-3 (Asp175) (5A1E, Cell Signaling, 1:1000). Subsequently, membranes were incubated for 1 hour with HRP-conjugated secondary antibodies (goat anti-mouse, Sigma, 1:50.000, or anti-rabbit, Cell Signaling, 1:5000). Signals were developed using ECL prime detection Kit (GE Healthcare) and visualized with FluorChemQ imaging system (Alpha Innotech, San Leandro, USA). Quantification was performed by Image J software.

The expression of CK 18 in the cytoplasm of biliary epithelial cells has been reported by Hayashi et al.^{23 (link)}. Therefore, we performed CK M30 immunostaining of a biliary ductal cancer specimen. A mouse monoclonal antibody (M30 CytoDEATH; Roche Applied Science, Mannheim, Germany) was used for M30 immunostaining^{35 (link)}.