Mica micb

1,000,000 G9_pCDH or 500,000 U1242_LRP cells/well were seeded in complete DMEM in 6-well plates in triplicates and immediately infected with CAN-2409 MOI 100/well. For all experiments, two biological replicates or a time course was performed. Treatment with GCV (10 μg/mL), AZD6738 (IC₅₀), AZD1390 (IC₅₀), or combination was performed 24 h after seeding. 24 and/or 48 h after treatment, cells were harvested, washed, and stained with the corresponding antibodies PD-L1 (Alexa Fluor 647, 750 R&D Systems FAB1561R-025, 5 μL/test) or MICA/MICB (clone 6D4 Alexa Fluor 647, PE/Cyanine7, Biolegend) and LIVE/DEAD Fixable Violet Dead Cell Stain Kit (#L34963, Thermo Fisher Scientific) and fixed using 4% PFA. For intracellular staining with γH2AX(Ser139) (clone 2F3, fluorescein isothiocyanate [FITC], Alexa Fluor 647 Biolegend), cells were first fixed with 4% PFA followed by antibody staining in permeabilization medium (Thermo Fisher Scientific) for 40 min at room temperature. Flow cytometry was performed with BD Fortessa or BD Aria, and 10,000 events were counted. Data were analyzed using FlowJo software (Bio-Rad).