Celltracker blue

For either CHO cells or Jurkat cells, 2–5 × 10⁷ cells were washed with 10 ml of PBS and resuspended in 35 ml of serum-free RPMI-1640 (Mediatech) containing 20 µM Cell Tracker blue (Life Technologies) and were incubated at 37°C for 40 min. The cells were washed with 5 ml of PBS followed by 5 ml of trypsin (Mediatech). The dyed cells were resuspended in 60 ml RPMI growth medium, consisting of RPMI-1640 medium (Mediatech) supplemented with 10% fetal bovine serum (FBS; Hyclone), 1× Pen/Strep, 1× Glutamax, 1× NEAA (all from Life Technologies), and incubated overnight at 37°C for use in GEMs the following day.

E-64 was obtained from Sigma-Aldrich. Caspase inhibitors were obtained from Enzo Life Sciences. Anti-paxillin (clone 349) was purchased from BD Biosciences. Anti-Pyk2 and anti-GAPDH were acquired from EMD Millipore. Anti-talin was obtained from Santa Cruz Biotechnologies. Anti-caspase-6 and anti-lamin were acquired from Cell Signaling Technology. Anti-mouse and anti-rabbit conjugated to HRP were from Jackson ImmunoResearch. Cell Tracker Blue, Cell Tracker Orange, A647-conjugated phalloidin and A488-conjugated anti-tubulin were obtained from Life Technologies. Recombinant human caspase-6 and the Z-VVR-AMC substrate were purchased from Enzo Life Sciences. The Z-Arg-Arg-pNA.2 HCl (Z-RR) substrate was purchased from Bachem. The FAM-FLICA caspase-6 assay kit was purchased from ImmunoChemistry Technologies. Transfection was carried out with the INTERFERin (Polypus) and caspase-6 siRNA (Santa Cruz Biotechnologies) or scramble siRNA (GE Dharmacon). Antibodies to EhCP1 and EhCP4, and WRR483 and WRR605 inhibitors were a gift from Dr. Sharon Reed, University of California, San Diego). Human IL-1β secretion in cell culture was quantified by ELISA (R&D Systems). LDH levels were measured using the CytoTox-ONE homogeneous membrane integrity assay (Promega).

RNK-16 cells were stained with 80 μM Cell Tracker Blue (CTB) (Molecular Probes) and were incubated at the indicated ratios with 3T3-E1A cells in 24-well plates for 6 h at 37 °C. After 6 h, all cells were stained with 100 nM TMRE (Molecular Probes). All cells were collected with trypsin-EDTA and processed for flow cytometry on a BD LSR. A non-blue gate was used to separate 3T3-E1A cells from CTB-RNK-16 cells for analysis of mitochondrial membrane potential of the 3T3-E1A cells. CTB-stained RNK-16 cells retained equivalent cytolytic activity as non-stained RNK-16 cells.

Cell growth medium, Dulbecco’s Minimum Essential Medium (DMEM) low glucose (Cat# D5523-10X1L), Penicillin G, Streptomycin, and Nystatin were all purchased from Sigma Chemicals Co. (St Louis, U.S.A.). CM-H2DCFDA, DiOC6, Mito-Tracker Green FM, Cell Tracker™ Red (CTR), Cell Tracker™ Blue (CTB), and CFSE were obtained from Molecular Probes (Eugene, U.S.A.). Matrigel (HC) was procured from BD Biosciences (Bedford, MA). Primary antibodies HK-II (Cat# SC-6521), PKM-2 (Cat# SC-65176), TFAM (Cat# SC-23588), Glut-1 (Cat# SC-7903), purchased from Santa Cruz Biotechnology (CA); HIF-1α (Cat# 3716S), SDH-A (Cat# 11998S), VEGF-R2 (Cat# 2479 S), MMP-9 (Cat# 13667S), AMPK-α (Cat# 2532S), p-AMPK-α (Cat# 2535 S), ACLY (Cat# 4332S), PGC-1α (Cat# 2178S), VDAC (Cat# 4866S) procured from Cell Signaling Technology (Danvers, MA, USA); and β-Actin (612657) obtained from BD Biosciences (CA), whereas Horseradish Peroxidase (HRP) conjugated secondary antibodies were procured from Thermo Fisher Scientific (USA).

For live cell imaging of in vitro Cre transfer, B16-GFP-Cre cells were added to adherent reporter MEF that had been labeled with 5 uM of CellTracker Blue (Molecular Probes #C2110) according to the manufacturer’s protocol. The video recording was initiated after 2 hours. Images were collected every 3–4 minutes with xyzt acquisition mode using an Axio Observer.Z1 microscope with the LSM 700 scanning module (Zeiss, Jena, Germany). Cultures were maintained at 37°C, 5% CO₂ using a Heating Insert P Lab-Tek S1 with an Incubator PM S1 (Zeiss, Jena, Germany).
For imaging of in vivo Cre transfer, B16-GFP-Cre tumors were grown in reporter mice as described above. After 18–20 days, mice were sacrificed, and tumors were harvested, coated in OCT, and flash frozen in liquid nitrogen. Cryosectioning was then performed to generate tumor sections 15 μm thick, which were then imaged with a Nikon D-Eclipse C1TE2000 confocal microscope (Nikon, Tokyo, Japan).

Intracellular reduced GSH was analyzed using 4‐chloromethyl‐6,8‐difluoro‐7‐hydroxycoumarin (Cell Tracker Blue; Molecular Probes, Eugene, OR, USA). Ten oocytes from each group were incubated in the dark in 10 μM CellTracker Blue for 30 min at 37°C. The oocytes were rinsed twice with PBS (−) containing 0.1% (w/v) polyvinyl alcohol (PVA). Oocyte GSH levels were measured using an inverted fluorescence microscope with UV filter (Blue image, 370 nm, BZ‐710, Keyence).
Intracellular ROS levels were measured using 2′7′‐dichlorodihydrofluorescein diacetate (CM‐H₂DCFDA; Molecular Probes, Eugene, OR, USA). Ten oocytes from each group were incubated in the dark in 10 μM CM‐H₂DCFDA for 30 min at 37°C. The oocytes were rinsed twice with PBS (−) containing 0.1% (w/v) polyvinyl alcohol (PVA). To analyze ROS levels, 2,7‐dichlorofluorescein (DCF) fluorescence signals were detected using an inverted fluorescence microscope (green channel: 480 nm, BZ‐X710, Keyence).
In measuring ROS and GSH levels, the average fluorescence intensity of fresh oocytes was set to 1.0, and the reported values represent fold differences in fluorescence intensity.