Figure 4.

Analyses of ROS production in the PMs of LTM and p190RhoGEF TG mice. (A) Images show LPS-mediated ROS production in PMs from LTM and TG mice. PMs (1 x10⁶) that were loaded with 5 μM DCFDA for 15 minutes were stimulated with LPS (1 μg/ml). Representative microscopic fields of DCF fluorescence from the cells after a 30-minute stimulation with either control (PBS) or LPS are shown. Scale bars = 50 μm. (B) The DCF fluorescence intensity of PMs from LTM and TG mice after a 30-minute stimulation with LPS is shown. The data are the averaged intensities from three microscopic fields (10 cells in each field). The data are presented as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparison tests. Significant comparisons are noted as *p < 0.05, **p < 0.01. The experiments were repeated three times. (C) LTM and TG PMs (2.5 x10⁵) were loaded with 5 μM DCFDA for 15 minutes and then stimulated with LPS (100 ng/ml) for 30 minutes. The DCF fluorescent cells were measured using flow cytometry. The numbers indicate the mean fluorescence intensity (MFI). The data shown are representative of at least five separate experiments. (D) The MFI of PMs from LTM and TG mice after a 30-minute stimulation with LPS is compared and the data are presented as the mean ± SEM. Statistical significance was analyzed by one-way ANOVA with Tukey’s multiple comparison tests (*p < 0.05, **p < 0.01). (E) LTM and TG PMs cultured in a black 96-well plate (1.5 x10⁵/well) were stimulated with LPS (100 ng/ml). Luminescence was detected 2 minutes after adding lucigenin (200 μM) for 60 minutes at 2-minute intervals. Experiments were repeated twice with similar results.