Fig 2: Inhibition of the NF-?B signaling pathway in HeLa cells by PA. Whole cell lysates were used to determine the expression levels of I?Ba, p-I?Ba (Ser32), NF?B p65, and p-NF?B p65 (Ser536) after treating cells with 10 mM and 20 mM PA for 48 h.

Fig 3: Effect of XDT-cs on the protein level of ICAM-1, MMP-9, COX-2, and p65 in glutamate-induced PC12 by WB assay. Representative bands are shown in (a). The levels of ICAM-1 (b) and MMP-9 (c), and COX-2 (d) and phosphorylation levels of p65 (e) were normalized to the control. Results were expressed as the mean ± SD (n = 3). **P < 0.01 vs. the control group; #P < 0.05 and ##P < 0.01 vs. the model group.

Fig 4: MyD88 and ERK1/2 Signaling Downstream of IL-33 Control the Development of Red Pulp Macrophages (RPMs)(A) Quantification of RPMs (percentage among CD11clow Ly6Glow NK1.1low SSC-Alow cells) in spleens of WT and Myd88-/- mice with or without treatment with hemin alone or IL-33 + hemin (one daily injection for 3 days; see STAR Methods).(B) Human macrophages (generated by culture of CD14+ monocytes from blood) treated for 5 min with phosphate-buffered saline (NT), hemin (40 µM), IL-33 (10 ng/mL), or a combination of hemin + IL-33, and assessed using a phospho-kinase array. pERK1/2 is shown in red boxes. Quantification of the intensity of ERK1/2 phosphorylation signal (pixel density) was performed using ImageJ. Data represent mean ± SEM and are representative of three independent experiments.(C) Mouse bone-marrow-derived macrophages of WT and Il1rl1-/- mice treated in vitro for 5 min with PBS (NT), hemin, IL-33, or a combination of hemin + IL-33, and assessed for pERK1/2, ERK1/2, and phospho-p65 (NF-?B) using western blotting. Semi-quantification of signal intensity (mean ± SEM) of ERK1/2 phosphorylation (pERK/Total ERK) using ImageJ yielded WT IL-33: 0.74 ± 0.02 versus WT IL-33+Hemin: 1.24 ± 0.09, p < 0.05. Semi-quantification of signal intensity (mean ± SEM) of p65 phosphorylation using ImageJ yielded WT IL-33: 11750 ± 3500 versus WT IL-33+Hemin: 12607 ± 4500, p = 0.09.(D and E) C57/Bl6 Spicigfp/igfp reporter mice were treated with ERK1/2 inhibitor U0126 or DMSO control for 6 weeks (see STAR Methods). ERK1/2 phosphorylation in splenic extracts (D), and splenic CD11blowSpic-EGFPhi RPMs and CD11b+Spic-EGFPint pre-RPMs (percentages among CD11clow Ly6Glow NK1.1low SSC-Alow cells) (E), were quantified at the end of the experiment. A control non-reporter untreated mouse (C57/Bl6) is included as control in (D).Each lane in (C) and (D) represents a separate mouse. Each dot in (E) represents a separate mouse. ****p < 0.001.

Fig 5: Effects of GBE on NF-?B binding activity and translocation. BV-2 cells were pre-treated with GBE (25 and 50 µg/ml) for 1 h, and then incubated with or without LPS (0.1 µg/ml) for 24 h. (A) The binding of NF-?B p65 and p50 to COX-2 promoter was analyzed using streptavidin-agarose pulldown assay. The protein levels of NF-?B p65 and p50 in whole-cell lysates (B) and nucleus (C) were measured by western blotting analysis. ß-actin and Lamin B1 were used as controls for sample loading. (D) The translocation of p50 and p65 from cytoplasm to nucleus were observed in BV-2 cells with indicated treatment by immunofluorescence imaging analysis. Confocal microscopy analysis showed that LPS could effectively promote p50/p65 entry into the nucleus, while GBE treatment significantly reduced their entry into the nucleus.