Skip to main content
. 2015 Dec 7;10(12):e0144229. doi: 10.1371/journal.pone.0144229

Fig 4. The effect of DYRK1A inhibition and downregulation is mediated via NFAT.

Fig 4

(A) To analyze the effect of DYRK1A inhibition on the amount of NFAT or NF-kB bound to the viral LTR, a ChIP-qPCR analysis was performed in TZM-bl cells treated with either 240 μM of INDY, the DMSO control, or 12.5 ng/ml TNFα. Sheared DNA was immunoprecipitated with either control IgG, anti-NFAT, or anti-NF-κB antibodies and levels of bound LTR DNA were analyzed by qPCR. (B) The effect of DYRK1A downregulation on LTR driven transcription in the presence of 10μM NF-kB inhibitor BAY or 300 ng/ml NFAT inhibitor FK506 (C) was analyzed by co-transfection of HEK293T cells in 96-wells plates with 5 ng of LTR-luciferase reporter construct and 12.5 ng, 25 ng or 50 ng of shDYRK1A or the shControl vector. Luciferase activity was analyzed 48-hours post-transfection as a measure for LTR activity and expressed relative to the shControl. Data is shown as mean and SD of three independent experiments. (D) Nuclear localization of NFAT was studied in TZM-bl cells cultured for 24 hours in the absence or presence of 240 μM INDY. Subsequently, cells were stained with Hoechst and anti-NFAT and analysed by confocal fluorescent microscopy. Results are representative of at least two independent experiments. Significance was determined with an unpaired student’s T test. *p<0.05, **p<0.01, ***p<0.001.