Figure 4.

Knockdown of ARID5B leads to a decrease in mitochondrial membrane potential, oxidative mitochondrial metabolism, and IFN-γ production. NK-92 cells were transduced with an empty control pLKO.1 vector or a pLKO.1 vector containing an ARID5B-specific shRNA. (a) Western blot of ARID5B and β-actin in the control and shARID5B NK-92 lines (left) and quantification by densitometry (right). (b) Representative FACS plots (left) and cumulative fold differences in MitoTracker MFI values (right) from each indicated NK-92 cell line. Experiments were replicated twice. (c) The indicated NK-92 cell lines were stained with DAPI and MitoTracker dyes and visualized by confocal microscopy. Shown are representative images (left) and cumulative MitoTracker intensity values calculated from 11 individual cells (right). Scale bars are 10 µm. Results are from two independent replicates, with similar results observed in both experiments. a.u., arbitrary units. (d) qRT-PCR was used to determine the ratio of mitochondrial DNA to genomic DNA for each indicated NK-92 cell line. Results are from three independent replicates. (e) OCR profiles of the control and shARID5B NK-92 cell lines in a representative experiment and averages for maximal respiration, ATP-linked respiration, and SRC. (f) ECAR profiles for the indicated NK-92 cell lines in a representative experiment and averages for maximal glycolysis and glycolytic reserve. All cumulative OCR and ECAR results were replicated twice. (g) Quantification of ATP in control and shARID5B cell lines. (h) Control and shARID5B NK-92 cells were cultured overnight with and without IL-12 and IL-18 before FACS analysis of NK cell functional readouts. Representative FACS plots and cumulative data of the frequencies of IFN-γ expression by NK-92 cells are shown. (g and h) Experiments were replicated twice. Error bars represent SEM. Paired Student’s t tests were used to determine statistical significance. *P < 0.05, **P < 0.01. n.s., not significant.