Figure 5

CD16+ monocytes are less protected from oxidative stress. (a) Supervised clustering of transcripts related to glutathione metabolism based on the normalized expression levels of individual transcripts using GEPAS. Red and blue color on the scale indicates high and low intensities, respectively. Validation of GPX1 and MGST1 expressions in isolated monocyte subsets by real-time PCR (b) and western blotting with β-actin as a loading control (c). The numbers below the western blots are relative intensity values after normalizing with the actin control. (d) Bar graph showing the ratio of GSH (i.e., reduced state) with respect to GSSG (i.e., oxidized state) in isolated monocyte subsets. (e) Bar graph depicts the percentages of apoptotic cells, that is, Annexin-V single-positive and Annexin-V/TO-PRO3 double-positive cells in the isolated monocyte subsets treated for either 2 or 4 h with the indicated concentrations of H₂O₂ with respect to their respective untreated monocyte subsets. (f) Bar graphs indicating the increase in the percentages of apoptotic CD16− monocytes that were either knockdown for GPX1 or MGST1 with respect to cells knockdown with siRNA control that were either left untreated or treated with the indicated concentrations of H₂O₂ for 4 h. Two different siRNA sequences for each target were used. Data are plotted according to the formula as shown. All data shown are from at least three different independent experiments and error bars represent S.E.M. ^** indicates P<0.01 and ^* indicates P<0.05