Caspase 7 standard

Apoptosis was induced in LOX-CXCL16, LOX-pcDNA and truncated LOX-ΔCXCL16 clones by the addition of 0.1 µg/mL camptothecin (Sigma-Aldrich, St. Louis, MO, USA) applied in a stock solution in DMSO, in the presence or absence of 50 ng/mL recombinant CXCR6 (BIOZOL, Eching, Germany). The final solvent concentration of 0.1% DMSO in camptothecin-treated cultures was also used in controls. After stimulation, cleavage of poly(ADP Ribose) polymerase (PARP) was measured by Western blot (150,000 cells/25 mm² flask, grown for 30 h and stimulated for 18 h) as described above using an antibody specifically detecting cleaved PARP (Asp124, 1:500, Cell Signaling Technology, Danvers, MA, USA). An antibody against GAPDH (1: 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) served as the loading control.
In T98G glioblastoma cells (250,000 cells/25 mm² flask, grown for 24 h), apoptosis was induced with 400 µg/mL TMZ (solved in DMSO) for 48 h in the presence or absence of 50 ng/mL recombinant CXCR6 (BIOZOL, Eching, Germany). Cells were lysed, and caspase 3/7 activity was measured as previously described [13 (link)] and normalized using a caspase 7 standard (Enzo Life Science, Lörrach, Germany).

Primary human meningioma cells were plated into 96-well dishes (10,000 cells/ well), grown for two days in 20 % FCS-supplemented DMEM, washed in 37 °C-thermostatted 0.5 % FCS-supplemented DMEM for three times (20 min, respectively), and stimulated in the same medium with 50 μg/ml camptothecin (Sigma Aldrich, Steinheim, Germany), 10 nM CXCL16 (Pepro Tech) or with combination of both for up to 24 h. In parallel, control wells without/with stimulation in DMSO were used. For detection of active caspase-3 amounts, samples were washed in PBS and incubated in 100 μl Homogeneous Caspase 3/7 substrate (Apo-ONE® Homogeneous Caspase-3/7 Assay; Promega, Madison, USA) for 30 min according to the manufacturer’s instruction and as described before [15 (link)]. The amounts of active caspase-3 were determined in relation to a caspase 7 standard (Enzo Life Science, Lörrach, Germany), and camptothecin-stimulated wells were set 100 %.

After RNAi silencing and TMZ exposure of T98G cells (described before) samples were washed in PBS and incubated in 100μl Homogeneous cCaspase-3/7 substrate (Apo-ONE® Homogeneous Caspase-3/7 Assay; Promega, Madison, USA) for 30 min according to the manufacturer's instruction and as described before [27 (link)]. The amounts of active Caspase-3/7 were determined in relation to a Caspase-7 standard (Enzo Life Science, Lörrach, Germany) as units/μl.