Nox inhibitors inhibit MK ploidy. (A) Nox inhibitor, DPI, decreases ROS production in MKs. The histogram depicts general ROS level detected by H₂DCF-DA in CD41-PE–positive cells of cultured BM, as described in “Methods.” DPI was added as indicated, and H₂O₂ was added as a positive control, all as described in “Methods.” (B) DPI decreases ploidy of MKs in BM culture. (Left) BM cells were cultured (+TPO) for 3 days with or without 0.2 μM DPI. Cells were then labeled with CD41-FITC antibody followed by PI staining before FACS analysis on a BD FACScan. (Right) Percentage of MKs undergoing S phase. Cell-cycling cells were determined by selectively gating the area between 2N and 4N DNA peaks, as shown in the histogram profiles. Data were analyzed with FlowJo software (TreeStar). Statistical analysis was applied with the use of the t test for paired values. No significant difference was found (P > .05). (C) Apocynin decreases ploidy of MKs in BM culture. BM cells were cultured (+TPO) for 3 days with or without 200 μM apocynin. (Left) Cells were then labeled and analyzed as described above. (Right) Percentage of MKs undergoing S phase. Cell-cycling cells were determined as described above. Statistical analysis was applied with the use of the t test for paired values. No significant difference was found (P > .05).