Figures & data
Figure 1. Effect of methoxychlor on FcϵRI-mediated β-hexosaminidase release from mast cells. β-Hexosaminidase release by (A) RBL-2H3 cells and (B) BMMC. Anti-DNP IgE-sensitized mast cells were treated with ethanol alone (control) or various concentrations of methoxychlor for 20 min. The cells were subsequently stimulated with DNP-HSA for 30 min and then β-hexosaminidase release was measured. (C) Viability of RBL-2H3 cells. Viability of cells first sensitized with anti-DNP IgE, then treated with various concentrations of methoxychlor or ethanol, and then stimulated with DNP-HSA, was assessed using WST-8 solution. Control cells were treated with antigen, but not with methoxychlor. Data shown are mean ± SD (n = 3). A Dunnett’s test was used to assess significance of difference versus control; *p < 0.05, **p < 0.01.
Figure 2. Effect of methoxychlor on calcium ionophore-induced β-hexosaminidase release from RBL-2H3 cells. RBL-2H3 cells were simultaneously treated with various concentrations of A23187 or with ethanol and with 20 μM methoxychlor for 30 min. β-Hexosaminidase release from the cells was then measured. Data shown are mean ± SD (n = 3). No statistical significance of the difference against the corresponding control was found when assessed using a Student’s t-test.
Figure 3. Effect of methoxychlor on PI3K inhibition by wortmannin in RBL-2H3 cells. Anti-DNP IgE-sensitized RBL-2H3 cells were incubated with various concentrations of wortmannin for 30 min. The cells were washed and treated with 20 µM methoxychlor for 20 min. The cells were subsequently stimulated with DNP-HSA for 30 min. Then, the β-hexosaminidase release from the cells was measured. Control cells were not treated with methoxychlor. Data shown are mean ± SD (n = 3). A Student’s t-test was used to assess the statistical significance of difference versus corresponding control; *p < 0.05.
Figure 4. Effect of methoxychlor on phosphorylation of PI3K in RBL-2H3 cells. Anti-DNP IgE-sensitized RBL-2H3 cells were treated with or without 20 μM methoxychlor for 20 min. The cells were subsequently stimulated with or without DNP-HSA for 30 min. Cell lysates were then prepared, and immunoblot analyses performed. The p-PI3K represents phosphorylated PI3K. (A) Representative image of PI3K phosphorylation from two independent immunoblot analyses. (B) Ratio of p-PI3K/PI3K relative to values for intact cells.
Figure 5. Effect of methoxychlor on phosphorylation of FcϵRI-mediated signaling factors in RBL-2H3 cells. Anti-DNP IgE-sensitized RBL-2H3 cells were treated with or without 20 μM methoxychlor for 20 min. Cells were subsequently treated with or without DNP-HSA for 30 min. Cell lysates were then prepared and immunoblot analyses performed. The p-Lyn, p-Syk, p-Btk, and p-PLC represent phosphorylated Lyn, Syk, Btk, and PLC, respectively. Representative image of phosphorylation of (A) tyrosine kinases and (B) PLCγ from three independent immunoblot analyses.
Figure 6. Effect of methoxychlor on IgE-mediated PCA in mice. Seven-week-old female BALB/c mice were intravenously injected with 200 µg DNP-HSA containing 0.5% Evans Blue dye 24 h after intradermal administration of anti-DNP IgE (20 ng) in PBS and PBS alone into, respectively, their right and left ears. Methoxychlor (23 mg/kg BW) in mineral oil or mineral oil alone (control) was orally administered 1 h before antigen treatment. (A) Photographs of representative right ears from methoxychlor-treated and control mice 30 min after administration of DNP-HSA. (B) Evans Blue dye was extracted from each ear and the absorbance measured at 620 nm (n = 6). The absorbance of the dye extracted from the left ear was subtracted from that from the right ear. A Student’s t-test was used to assess the significance of the difference against control.
