Figure 7. VLCs express ARG1 but not iNOS.

(A) CD11c-isolated VLCs from ID8 ascites were analyzed for iNOS expression by Western blot. iNOS expression was observed in LPS-stimulated BMDCs (positive control; lane 1) but not in 1×10⁵, 5×10⁴, and 1×10⁴ VLCs (lanes 2–4, respectively). Calreticulin was used as a loading control (bottom panel). (B–C) Analysis of ROS production by VLCs. (B) CD11c⁺ VLCs express ROS, as indicated by the fluorescence in cells incubated with the ROS-indicator DCFDA (open black line) in comparison to cells analyzed in the absence of DCFDA (shaded line). (C) To assess the specific contribution of ARG1 to VLC ROS production, VLCs were incubated with DCFDA in the presence of the indicated concentration of the ARG1 inhibitor nor-NOHA. Cells were incubated for 1 hr at 37°C, and DCFDA fluorescence was analyzed by FACS. The standard deviation from three independent experiments is shown. (D) Human ovarian cancer CD11b⁺ cells (lane 1) or naïve blood monocytes (lane 2) were analyzed by western blot for arginase 1 expression (bottom panel); gp96 was used as loading control (top panel).