Figure 1.
TAK-981 treatment enhances M1 polarization and NK activation in an IFN1-dependent manner. (A) Amount of IFNβ and IP-10 protein in the supernatant of 1 μM TAK-981- or 10 kU/mL IFNα2-treated hMDM at indicated time points (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (B) Western blots of indicated proteins in TAK-981–treated hMDM at 24 hours. (C) Representative flow cytometry histogram and median fluorescence intensity (MFI) of indicated proteins in 50 ng/mL IFNγ (M1 polarizing stimulation)-, 20 ng/mL IL-4 (M2 polarizing stimulation)-, and/or 1 μM TAK-981–treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (D) MFI of indicated proteins in 20 μg/mL anti-IFNAR2 antibody (aIFNAR2)-, 1 μM TAK-981-, and/or 10 kU/mL IFNα2-treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (E) mRNA expression of indicated genes in 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated hMDM at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (F) Representative flow cytometry histogram and MFI of CD206 in 20 ng/mL IL-4- and/or 1 μM TAK-981–treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (G) mRNA expression of CCL22 in 20 ng/mL IL-4- and/or 1 μM TAK-981–treated hMDM at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (H) mRNA expression of IP-10 and CD69 in 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated human NK cells at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (I) Representative flow cytometry histogram and percentage of CD69+ cells or MFI of CD69 in 1 μM TAK-981- or 10 kU/mL IFNα2-treated human NK cells at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). *P < .05, **P < .01, ***P < .001, ****P < .0001. Experiment was repeated using hMDM or human NK cells generated from at least 2 healthy PBMC donors. mRNA, messenger RNA; N.S., not significant (P > .05); PBMC, peripheral blood mononuclear cells; SD, standard deviation.

TAK-981 treatment enhances M1 polarization and NK activation in an IFN1-dependent manner. (A) Amount of IFNβ and IP-10 protein in the supernatant of 1 μM TAK-981- or 10 kU/mL IFNα2-treated hMDM at indicated time points (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (B) Western blots of indicated proteins in TAK-981–treated hMDM at 24 hours. (C) Representative flow cytometry histogram and median fluorescence intensity (MFI) of indicated proteins in 50 ng/mL IFNγ (M1 polarizing stimulation)-, 20 ng/mL IL-4 (M2 polarizing stimulation)-, and/or 1 μM TAK-981–treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (D) MFI of indicated proteins in 20 μg/mL anti-IFNAR2 antibody (aIFNAR2)-, 1 μM TAK-981-, and/or 10 kU/mL IFNα2-treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (E) mRNA expression of indicated genes in 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated hMDM at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (F) Representative flow cytometry histogram and MFI of CD206 in 20 ng/mL IL-4- and/or 1 μM TAK-981–treated hMDM at 48 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (G) mRNA expression of CCL22 in 20 ng/mL IL-4- and/or 1 μM TAK-981–treated hMDM at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (H) mRNA expression of IP-10 and CD69 in 20 μg/mL aIFNAR2- and/or 1 μM TAK-981–treated human NK cells at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). (I) Representative flow cytometry histogram and percentage of CD69+ cells or MFI of CD69 in 1 μM TAK-981- or 10 kU/mL IFNα2-treated human NK cells at 24 hours (mean with SD; n = 3 biological replicates, 2-tailed unpaired Welch’s t test). *P < .05, **P < .01, ***P < .001, ****P < .0001. Experiment was repeated using hMDM or human NK cells generated from at least 2 healthy PBMC donors. mRNA, messenger RNA; N.S., not significant (P > .05); PBMC, peripheral blood mononuclear cells; SD, standard deviation.

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