Figures & data
Table 1. Animal group and immunogens
Figure 1. HAV-specific antibody (IgG) titers. Female ICR mice were divided into 5 groups (8/group) and were subcutaneously immunized with Physiological saline, HAV or HAV mixed with the selected dose of Kyn (except for the control group), as described in the experimental procedures listed in . The serum samples were collected and analyzed using ELISA on the 4th (A), 8th (B), 12th (C), and 16th (D) week. The Y value is the log value of the endpoint titers. Blank, blank control group; vector, negative control group; HAV, HAV group; LA, HAV co-administrated with low dose adjuvant group; HA, HAV co-administrated with high dose adjuvant group.
Figure 2. Effect of Kyn on LPS-induced IgM secretion in B cells. FCM was performed to determine B cell purity. (A) Before separation, (B) after separation. B cells were stimulated with the indicated combination of LPS (10 μg/mL) and selected concentrations of Kyn. Supernatants were collected after 72 h and analyzed for IgM using ELISA, as described in the experimental procedures (C). The experiment was repeated 3 times.
Figure 3. Effect of Kyn on miR30b expression level. Real-time PCR was used to assay the miR30b expression level (A) in B cells treated with the indicated combination of LPS (10 μg/mL) and Kyn (100 μM) at 8 h. The results represent 3 separate experiments. The products of real-time PCR were evaluated by agarose gel electrophoresis (B); line 1, 20bp ladder; line 2, miR30b; line 3, U6 snRNA; and sequencing (C).
Figure 4. Involvement of miR30b in Kyn-mediated suppression of IgM responses induced by LPS in B cells. Supernatants were harvested from the B cells incubated with LPS (10 μg/mL), LPS (10 μg/mL) + miR30b, LPS (10 μg/mL) + miR30b inhibitor, LPS (10 μg/mL) + Kyn (100 μM), LPS (10 μg/mL) + Kyn (100 μM) + miR30b, or without at 72 h. The effect of miR30b on the generation of IgM (A) or on kyn-mediated suppression of IgM responses (B) was assayed using ELISA. The experiment was repeated 3 times.
Figure 5. Bach2 is a novel target of miR30b. The interaction sites predicted between miR30b and Bach2 is shown in (A). Agarose gel electrophoresis assayed related fragments, plasmids involving in the recombinant plasmids, pmirGLO-hB and pmirGLO-hB mt (B); line 1, DL 1000 Marker; line 2, objective fragment of wild type; line 3, forward objective fragment of mutant type; line 4, reverse objective fragment of mutant type; line 5, completely objective fragment of mutant; line 6, pmirGLO vector; line 7, pmirGLO-hB; line 8, double enzyme digestion of pmirGLO-hB; line 9, pmirGLO-hB mt; line 10, double enzyme digestion of pmirGLO-hB mt; line 11, DL15000 Marker. Objective fragments in pmirGLO-hB and pmirGLO-hB mt was evaluated using sequencing (C). Luciferase activity was detected using a dual-luciferase assay system at 48 h after transfection (D) and repeated 3 times. The underline indicates the mutant site in this Figure.
Figure 6. The effect of miR30b on Bach2 mRNA and protein levels. B cells incubated with miR30b, LPS (10 μg/mL), LPS (10 μg/mL) + Kyn (100 μM), LPS (10 μg/mL) + Kyn (100 μM) + miR30b, or without were harvested at 72 h. The non-treated B cells are considered the control. The Bach2 mRNA and protein levels were measured using real-time PCR and western blot, respectively. Kyn changed the Bach2 mRNA (A) and protein (C) levels in B cells. In the process of Kyn-mediated suppression of IgM responses induced by LPS, the effect of miR30b on Bach2 mRNA and protein levels is presented in panels (B) and (D), respectively. The results represent the mean of 3 separate experiments.
