Skewed hematopoietic differentiation in CAV-1 KO BM. WT or CAV-1 KO mice under normoxia or 3 weeks of hypoxia were used. BM was isolated from femora and tibiae; mature red blood cells were lysed, and remaining cells were stained for delineation of HSC differentiation using flow cytometry. (A) Gating strategy for HSC and progenitors. Time gating was used to check for and exclude fluidic disturbances in the flow cell. Aggregates were excluded based on blue laser forward and side light scatter (FSC and SSC, respectively) peak height, area, and width. Dead cells were gated out using a UV live/dead stain. Cell debris was eliminated on an FSC/SSC plot. Lin^neg/low cells were selected and further gated for primitive and multipotent progenitor cells (KSL) and committed myeloid progenitors (KL) based on standard c-Kit and Sca-1 expression patterns. HSC, MMP, and lymphoid-primed multipotent progenitor (LMPP) subsets were gated in KSL population based on CD135 expression levels. KL population was delineated into CMPs, GMPs, and MEPs based on CD34 and CD16/32 expression. FMO controls were used to set boundaries for stem and progenitor cell gates. A schematic representation of the mouse hematopoietic differentiation path is shown. (B-E) Box plots depicting the percentage of KSL, HSC, MMP, and LMPP subsets in WT and CAV-1 KO mice, with and without hypoxia exposure. (F-I) Box plots showing the percentage of KL, CMP, GMP, and MEP subsets in WT and CAV-1 KO mice, with and without hypoxia exposure. Data from 5 male mice in each group are shown. ANOVA values show differences across groups. Symbols represent significant differences between 2 groups (P < .05): #WT normoxia vs CAV-1 KO normoxia; ∼WT hypoxia vs CAV-1 KO normoxia; §WT normoxia vs CAV-1 KO hypoxia; *WT normoxia vs WT hypoxia; ¶WT hypoxia vs CAV-1 KO hypoxia; ¤CAV-1 KO normoxia vs CAV-1 KO hypoxia. FSC, forward scatter; SSC, side scatter.