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. 2012 Apr;86(8):4566–4577. doi: 10.1128/JVI.05545-11

Fig 3.

Fig 3

VPA suppression of IFN-γ production is associated with STAT5/T-BET inhibition. (a) Enriched human NK cells were cultured overnight with IL-12 plus IL-15 or IL-12 plus IL-18 in the presence or absence of VPA. The following day, NK cells were collected and mRNA was isolated. Following mRNA conversion to cDNA, the expression of IFN-γ was evaluated. Statistically significant values for VPA inhibition of IFN-γ transcript in primary NK cells costimulated with IL-12 and IL-15 or IL-12 and IL-18 were P < 0.01 for IL-12 and IL-15 costimulation and P < 0.05 for IL-12 and IL-18 costimulation. P values were calculated using Student's t test. (b) NK cell line NK-92 was cultured overnight with various combinations of cytokine stimulants in the presence or absence of VPA. The following day, secreted IFN-γ was quantified using ELISA. P values were calculated using Student's t test. (c) Enriched human NK cells from three separate donors were cultured overnight with various combinations of cytokine stimulants in the presence or absence of VPA. The following day, secreted IFN-γ was quantified using ELISA. A statistically significant value for VPA inhibition of IFN-γ protein secretion by primary NK cells costimulated with IL-12 and IL-15 or IL-12 and IL-18 was P < 0.05. P values were calculated using Student's t test. (d) Enriched primary human NK cells were cultured overnight with various combinations of cytokine stimulants in the presence or absence of VPA. The following day, extracted protein was evaluated by Western blotting for STAT5 phosphorylation (90 kDa [upper band]) or T-BET expression (62-kDa band). Actin was used as a loading control. Staining intensity was quantified with densitometry, and values are presented under the respective bands. (e) STAT5 knockdown was confirmed by real-time RT-PCR using NK-92 cells expressing STAT5-shRNA compared to the cells expressing the empty vector control (STAT5a, top left; STAT5b, top right). The STAT5-shRNA knockdown cell line or NK-92 cells expressing the empty vector control were used to determine IFN-γ inhibition by VPA in the presence of costimulation with IL-12 and IL-18 (bottom). P values were calculated using Student's t test. *, P < 0.05; **, P < 0.01. Error bars represent ± standard deviations.