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. 2018 Jul 16;2(14):1664–1679. doi: 10.1182/bloodadvances.2017013433

Figure 4.

Figure 4.

TNP-470–treated DCs trigger NF-κB transactivation of IL-12 and potentiate p38/JNK activation. (A) TNP-470/vehicle-treated DCs were stimulated with different TLR agonists (5 nM, 16hr). **P < .01 TNP-DC vs CTR-DC upon agonist stimulation. Data presented as mean ± SEM from 3 independent experiment. (B) TNP-470/vehicle-treated DCs were stimulated with LPS (1 µg/mL) for 0, 30, and 60 min. Total protein lysate was blotted with antibodies against p38, phosphor-p38, JNK, phosphor-JNK and GAPDH. Representative images from 2 independent experiment were showed. (C) TNP-470/vehicle-treated DCs were stimulated with LPS for 24 hours in the presence of 2 µM and 10 µM p38 inhibitor SB203580 and MAPK inhibitor PD98059. IL-12 secretion was detected by cytokine ELISA. ***P < .001 compared with TNP-470–treated DCs without inhibitor. Data presented as mean ± SEM from 2 independent experiment. (D) TNP-470/vehicle-treated DCs were stimulated with LPS (200 ng/mL) for 24 hours in the presence of 2 µM and 10 µM NF-κB inhibitor CAPE. IL-12 secretion was detected by cytokine ELISA. ***P < .001 compared with TNP-470–treated DCs without inhibitor. Data presented as mean ± SEM from 2 independent experiment. (E) Fold-of-change in expression of different NF-κB subunits, p52, p65, c-Rel and relB, which were normalized with GAPDH, in TNP-470/vehicle-treated DCs (200 ng/mL LPS for 4 hours). *P < .05, **P < .01 and ***P < .001 TNP-DC vs CTR-DCs. Data presented as mean ± SEM from 2 independent experiment. (F) TNP-470/vehicle-treated DCs was stimulated with LPS for 30 minutes. Expression of cRel, p65 and histone H3 (loading control) from nuclear fractions were detected. Representative images from 2 independent experiment were showed. (G) NF-κB p65 translocation assay. TNP-470/vehicle-treated DCs were stimulated with LPS for 30 minutes. The subcellular localization of p65 was visualized by immunofluorescence using anti-p65 antibody (red) and nuclei were stained by DAPI (blue). Representative images from 3 independent experiment were showed. Original magnification ×40. (H) Recruitment of NF-κB subunit c-Rel to IL-12 promoter. TNP-470/vehicle-treated DCs were stimulated with LPS for 0 and 2 hours. Cells were then cross-linked and lysed. DNA was immunoprecipitated with anti-cRel and control IgG. Degree of c-Rel recruitment to IL12 promoter was quantified by real time PCR. *** P < .001 TNP-DC vs CTR-DC. Representative data presented as mean ± SEM from 2 independent experiment.