Fig. 3

Neo-2/15 augments the expression of nutrient transporters and OXPHOS of CAR-NK cells. a, b Immunoblot for JAKs/STATs and Akt/mTOR pathways in BBζ stimulated with IL-2 or Neo-2/15 in the absence (a) or presence (b) of AsPC-1 cells. c The mRNA (top) and protein (bottom) levels of c-Myc in Neo-2/15 stimulated BBζ pretreated with Stafia1 (22 μM), AZD5363 (8 nM) or Rapamycin (10 nM) alone or in combination in the same concentration overnight were evaluated. Western blots are representative of three independent experiments. Data are presented as mean ± SD (ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 compared with DMSO group, ^##P < 0.01, ^###P < 0.001 compared with Stafia1 group, n = 3). d Heatmap showing the cytolytic activity against MSLN positive tumor cells of Neo-2/15 stimulated BBζ pretreated with Fludarabine (50 μM), NSC74859 (86 μM), Stafia1 (22 μM), AZD5363 (8 nM), Rapamycin (10 nM), 10058-F4 (60 μM), Nifuroxazide (3 μM), Stafia1 (22 μM) + AZD5363 (8 nM), or Rapamycin (10 nM) + Stafia1 (22 μM) overnight measured by the LDH assay. e ATP generation in BBζ stimulated with IL-2 or Neo-2/15 without (left) or with (middle) co-cultured with AsPC-1 cells for 4 hours or in Neo-2/15 stimulated BBζ pretreated with 10058-F4 (60 μM) for 30 minutes and then co-cultured with AsPC-1 cells for 4 hours (right). Data are presented as mean ± SD (***P < 0.001, n = 5). f Representation of pathways altered in IL-2 or Neo-2/15 stimulated BBζ based on transcriptome data. Pathways of interest are highlighted. g OCR was assessed using a seahorse analyzer in BBζ stimulated with either Neo-2/15 or IL-2 after co-culturing with AsPC-1 cells. h OCR was assessed in Neo-2/15 expanded BBζ pre-treated with or without 10058-F4 (60 μM) and subsequently co-cultured with AsPC-1 cells. i mRNA (top) and protein (bottom) levels of the nutrient transporters in Neo-2/15 or IL-2 stimulated BBζ without or with co-cultured with AsPC-1 cells. Blots are representative of three independent experiments. Data are presented as mean ± SD (**P < 0.01, ***P < 0.001, n = 5). j mRNA (top) and protein (bottom) levels of the nutrient transporters in Neo-2/15 stimulated BBζ pretreated with Stafia1 (22 μM), AZD5363 (8 nM) or 10058-F4 (60 μM) for 30 minutes after co-cultured with AsPC-1 cells. Blots are representative of three independent experiments. Data are presented as mean ± SD (ns, not significant, *P < 0.05, ***P < 0.001 compared with DMSO group, ^##P < 0.01, ^###P < 0.001 compared with Stafia1 group, n = 5)