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. 2022 Jun 13;12(6):e901. doi: 10.1002/ctm2.901

FIGURE 4.

FIGURE 4

Western blotting analysis of signalling pathway molecules involved in PD‐L1 expression on NK‐92, CAR NK‐92 and C4‐2 cells when cocultured with each other. (A–E) Representative and summary data (n = 3) of PD‐L1 (A, B), p‐PI3K (A, C), p‐AKT (A, D) and phosphorylated mammalian target of rapamycin (p‐mTOR) (A, E) protein levels in a panel of CAR NK‐92 cells, cocultured CAR NK‐92 cells, blocked CAR NK‐92 cells (treated with NKG2D blocker), NK‐92 cells, cocultured NK‐92 cells and blocked NK‐92 cells (treated with NKG2D blocker). The NKG2D blocker was added to effector cells and incubated for 1 h at 37°C prior to coculture with C4‐2 cells. (F–I) Representative and summary data (n = 3) of p‐AKT (F, G), p‐mTOR (F, H) and PD‐L1 (F, I) protein levels in a panel of CAR NK‐92 cells, cocultured CAR NK‐92 cells, blocked CAR NK‐92 cells (treated with wortmannin), NK‐92 cells, cocultured NK‐92 cells and blocked NK‐92 cells (treated with wortmannin). Effector cells were cocultured with C4‐2 cells in the absence or presence of the PI3K‐specific inhibitor wortmannin (10 μmol/L). (J–M) Representative and summary data (n = 3) of the protein levels of p‐PI3K (J, K), p‐mTOR (J, L) and PD‐L1 (J, M) in a panel of CAR NK‐92 cells, cocultured CAR NK‐92 cells, blocked CAR NK‐92 cells (treated with afuresertib), NK‐92 cells, cocultured NK‐92 cells and blocked NK‐92 cells (treated with afuresertib). Effector cells were incubated with C4‐2 cells in the absence or presence of the AKT inhibitor afuresertib (10 μmol/L). (N–Q) Representative and summary data (n = 3) of JAK/STAT pathway intermediaries, including p‐JAK1 (N, O), p‐JAK2 (N, P) and p‐STAT1 (N, Q) in C4‐2 cells cocultured with CAR NK‐92 cells compared with C4‐2 cells without any process. CAR NK‐92 or NK‐92 cells were cocultured with C4‐2 cells for 24 h and then harvested for western blotting. Bars represent the means ± SD, ANOVA followed by a Tukey post hoc test was used for multiple group comparisons (B–E, G–I, K–M), and Student's t‐test was used for two‐group comparisons (O–Q). *p < .05; NS, not significant