7 add

Tumors cells that were incubation with GNLY from HIS mice serum or isolated from tumor-bearing HIS mice were performed by staining annexin-V-PE and 7-ADD (BD Biosciences) according to the manufacturer’s instructions and then analyzed for apoptosis by flow cytometry. The tumor cells isolated from HIS mice were analyzed the expressions of cleaved caspase3, caspase7 and PARA by western blotting.

Metastatic CRC cells at confluence were detached by trypsinization and incubated with FITC-conjugated Annexin V (5 µL/5 × 10⁵ cells) (Annexin V apoptosis Detection Kit, Invitrogen, Carlsbad, California, USA) and with 7-ADD (5 µL/5 × 10⁵ cells) (BD Pharmingen, San Diego, CA, USA). Metastatic CRC cells maintained in the absence and presence of NaHS (100 µM, 72 h) were incubated for 15 min at resting temperature and then analyzed by Beckman Coulter Navios according to manufactures’ instructions.

LSPCs were isolated as described previously (McQualter and Bertoncello, 2015 (link); Rabata et al., 2017 (link)). Briefly, the lungs were chopped up using scalpels, and the resulting mince was digested by Liberase (0.048 mg/ml; Roche) and incubated for 45 min at 37°C with shaking. The suspension was passed through 18G and 21G needles and treated with DNase I (20 U/ml). Next, the suspension was passed through a 100 μm cell strainer, treated with a red blood cell lysis buffer, and passed through a 40 μm cell strainer. The resulting single-celled suspension was centrifuged to collect the cells. The pellet was suspended in a blocking buffer [1% BSA in HBSS (both Merck)] and incubated for 20 min at room temperature. After centrifugation, the cells were resuspended at 1 × 10⁷ cells/ml in a FACS buffer containing the selection antibody cocktail (anti-CD104, anti-EpCAM, anti-CD24, anti-CD49f, anti-CD45; see Supplementary Table 1) and incubated in ice in the dark for 20 min. The cells were washed with HBSS, passed through a 30 μm cell strainer, and incubated with 10 μl/ml 7-ADD (BD Biosciences) for 5 min in ice in the dark. Cells were then sorted using FACSAria II SORP (BD Biosciences).

Cells were treated with indicated concentrations of GZD824 or DMSO for 48 h at 37℃. After incubation, cells were collected and washed twice with pre‐cold PBS. About 6 × 10⁵ cells were resuspended in 100 μl of 1× BD binding buffer solution (#556454, BD), and then stained with 7‐ADD (#559925, BD) and Annexin V‐PE (#556422, BD) in the dark for 15 min. Finally, 400 μl of 1×BD binding buffer solution were added to stop dyeing. The cells were then measured on a Guava easyCyte flow cytometer (Merck, USA).