Cytoflex lx analyzer

DR-GFP plasmids were gifts from Maria Jasin (Memorial Sloan Kettering Cancer Center). MEFs were transfected with DR-GFP plasmids or plasmids expressing an intact GFP protein. Forty-eight hours after transfection, cells were infected with adenovirus expressing I-SceI. After 24 h, GFP positive cells were analyzed by flow cytometry using a Beckman CytoFLEX LX Analyzer. HR efficiency were normalized by transfection efficiency marked by GFP only cells.

AAVR-silenced HEK293T cells were used for AAVR mutant transfection and infected with wt AAVs for reporter assays. Mutants in the PKD1 or PKD2 domains were introduced into the eukaryotic expression vector pcDNA3.1(+)-AAVR by the Fast mutagenesis system (TransGen Biotech, China). At 24 h prior to transfection, cells were seeded into 96-well or 12-well plates at a density of 2.5 × 10⁴ cells per well or 3 × 10⁵ cells per well and transfected at 70% confluency using PEIMAX. A total of 100 ng plasmids was used for each well of the 96-well plate, or a total of 1000 ng plasmids was used for each well of the 12-well plate. At 8 h post-transfection, the medium was changed to AAV1-mCherry- or AAV5-mCherry-containing medium at a MOI of 5 × 10⁵ or 3 × 10⁶ vg cell⁻¹. At 48 h post-transduction, mCherry-positive cells were detected by flow cytometry by using a CytoFLEX LX analyzer (Beckman, USA). All primer sequences are listed in Supplementary Table 9.

For cell culture analysis, cells with MTSS1 or AIP4 knockdown were cultured, suspended, and stained with anti-human CD274-PE or control antibody for 45 min at 4 °C. After washing three times with PBS, cells were analyzed on a Gallios analyzer (Beckman Coulter Life Sciences). For tumor analysis, tumors were excised from euthanized mice, cut into pieces, and then digested with buffer (RPMI 1640 containing 2.5 mg/mL Dispase II, 2.5 mg/mL collagenase IV, and 50 µg/mL DNAse I) at 37 °C for 30–60 min. The cell suspension was filtered with a 70-µm strainer before erythrocyte lysis. The cells were cultured in RPMI-1640 with 10% FBS and Cell Stimulation Cocktail (plus protein transport inhibitors) at 37 °C for 4 h. The samples were incubated with a buffer mix (PBS containing 10% FBS, 1 µL CD16/CD32 Fc blocking antibody, and 0.1 µL Fixable Viability Dye eFluor™ 780) at 4 °C for 15 min and then incubated with CD45, B220, CD11B, CD3, and CD8 antibodies at 4 °C for 30–45 min. Granzyme B was detected by intracellular staining. Finally, the cells were analyzed on CytoFLEX LX analyzer (Beckman Coulter Life Sciences). The gating strategy is shown in Supplementary Fig. S2d. Data were analyzed with FlowJo v.10 (FlowJo LLC). The antibodies used in this study and their sources are listed in Supplementary Table S3.