Differential expression profiles of HO-1 during different stages of hematopoietic development and changes in levels of intracellular ROS, p38MAPK phosphorylation, and p21 expression in HO-1–deficient mice under steady-state conditions. Representative FACS plots of the levels of intracellular HO-1 protein (A), ROS (B), and p38MAPK phosphorylation (C) in whole BM, HSCs, MPPs, and restricted progenitors. BM cells from HO-1^+/+ and HO-1^+/− mice (n = 6 for each group) were harvested and stained with antibodies for phenotypically defined HSC (CD150⁺CD48⁻CD244⁻), MPP (CD244⁺CD48⁻CD150⁻), and more restricted progenitor (CD48⁺CD244⁺CD150⁻) populations. To assess intracellular ROS levels, stained cells were incubated with DCF, a fluorescent dye for detection of intracellular ROS. For measurement of intracellular HO-1 protein, and p38MAPK phosphorylation, stained cells were fixed, permeabilized, and then stained with antibodies against HO-1, and phosphorylated p38MAPK, respectively. The broken line in the first panel of Figure 1A is the isotope control with FITC-conjugated anti–mouse IgG antibody. Values are the mean percentages indicating the gated proportion of cells. Please note that some of the histograms were skewed to the left due to the number of events on the y-axis. p21 expression was assessed by real-time PCR in HO-1–deficient cells. KTLS HSCs or Lin⁻ BM cells were isolated from HO-1^+/− or HO-1^+/+ mice at steady state, total RNA was prepared from these cells for p21 mRNA quantitation using real-time PCR. p21 mRNA levels were accumulated in purified KTLS HSCs (displayed is an average fold change of four experiments) (D) but decreased in HO-1^+/− Lin⁻ BM cells (mean ± SD, n = 4) (E). The frequency of KTLS HSCs was analyzed from nucleated BM cells and are displayed as the mean (n = 3) (F).