3-(5-Hydroxyphenyl)-5-Phenyl-2-Pyrazolines as Toll-Like Receptor 7 Agonists

Figure 4

TLR7 agonist activity of compound 6. (a) Images of SEAP assay plates. THP1 cells were treated with 40 μM compound 6 for different durations. Whole-cell lysates were immunoblotted with antibodies against phospho-IRAK4 (Thr345/Ser346), −IKKα/B (Ser176/180), −IκBα (Ser32), and -p65/RelA NF-κB (Ser536). GAPDH level was used as an internal control. (b) Protein band intensities were measured using Image J software. The data are presented as means ± SD (n = 3). ns: not significant, , and . (c) THP1 cells were treated with 40 μM compound 6 for 10 min and then incubated with an antibody against p-p65/RelA (Ser536) or α-tubulin for 2 h followed by the addition of Alexa Fluor 555-(red fluorescence for p-p65) or Alexa Fluor 488-(green fluorescence for α-tubulin) secondary antibody for an additional 30 min. Nuclear DNA was stained with 0.1 μg/mL Hoechst 33258 (blue fluorescence) for 10 min. Fluorescent cells were captured using an EVOSf1® fluorescence microscope. Bars, 10 μm.

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