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. 2018 Sep 3;215(9):2379–2395. doi: 10.1084/jem.20172168

Figure 6.

Figure 6.

Overexpression of ARID5B results in increases in mitochondrial membrane potential, oxidative mitochondrial metabolism, and IFN-γ production. NK-92 cells were transduced with a control pCDH vector containing GFP or a pCDH vector containing GFP and ARID5B variant 2. (a) Western blot of ARID5B variant 2 and β-actin in each sorted GFP+ NK-92 cell line (left) and image quantification by densitometry (right). (b) Mitochondrial membrane potentials were determined by MitoTracker staining and FACS analysis in sorted GFP+ cells from the control and ARID5B variant 2 overexpression NK-92 lines. Cumulative data of the relative fold difference in MitoTracker staining between vectors are shown (right). Experiments were replicated twice. (c) Primary NK cells were transduced with the control or the ARID5B variant 2 overexpression vectors. Cells were analyzed for GFP expression and mitochondrial mass 72 h after transduction. Shown are representative FACS plots of GFP against forward scatter (FSC) in gated NK cells and MitoTracker histogram plots (left). Cumulative data from four donors of the relative fold difference in MitoTracker staining between vectors are also shown (right). Results are from two independent experiments. Similar results were observed in both experiments. (d) OCR profiles of the control and ARID5B variant 2–overexpressing NK-92 cell lines in a representative experiment and averages for maximal respiration, ATP-linked respiration, and SRC. (e) ECAR profiles for the indicated NK-92 cell lines in a representative experiment and averages for maximal glycolysis and glycolytic reserve. OCR and ECAR experiments were replicated twice. (f) Quantification of ATP in control and ARID5B-overexpressing cell lines in three independent replicates. (g) Control and ARID5B-overexpressing NK-92 cells were cultured overnight with and without IL-12 and IL-18 before FACS analysis. Representative FACS plots and cumulative data of the frequencies of IFN-γ expression by NK-92 cells are shown. Experiments were replicated twice. Error bars represent SEM. Paired Student’s t tests were used to determine statistical significance. *P < 0.05. n.s., not significant.