Figure 4

PiT1-deficient cells show impaired ROS production. (A,B) Measurement of intracellular ROS production upon LPS treatment using CM-H2DCFDA probe. (A) Flow cytometry analysis of CM-H2DCFDA fluorescence in BMDMs from Mx1-Cre; Pit1^lox/lox, i.e., PiT1-deficient cells (white bars) and controls (black bars) following 7-day differentiation. Macrophages were treated with complete medium with or without 1000 ng/ml LPS for 20 h. Each bar represents means ± S.E.M. of three independent experiments including BMDMs from at least three Mx1-Cre; Pit1^lox/lox and three control mice. ^#Indicates comparison to untreated cells; ^##p < 0.01, ^###p < 0.001; *indicates p < 0.05 as compared to control cells. Student’s unpaired t-test or an Unpaired t-test with Welch correction for groups with unequal variance was performed (B) Data from a representative CM-H2DCFDA fluorescence analysis. (C,D) Measurement of ROS production by luminol-amplified chemiluminescence. (C) BMDMs from Mx1-Cre; Pit1^lox/lox, i.e., PiT1-deficient cells (white bar) and control mice (black bars) following 7-day differentiation were harvested in HBSS and stimulated with fMLF at a final concentration of 10⁻⁵ M in presence of luminol and peroxidase. Chemiluminescence was directly quantified and given as the ratio between fMLF-treated and basal conditions. Data are means ± S.E.M of at least three independent experiments. Student’s unpaired t-test or an Unpaired t-test with Welch correction for groups with unequal variance was performed *indicates comparison with control cells; ***p < 0.001. (D) Representative chemiluminescence experiment.