Figure 1.
Anaerobic atmospheres inhibit cell proliferation.
Cells were incubated under aerobic and two types of anaerobic atmosphere conditions, N2A or COA. Cells were counted daily by hemocytometer. Air •–––•; COA ▪–––▪; N2A ▴----▴ . Data represent mean ± SD of 4 independent experiments.
Figure 2.
COA but not N2A preserves cell viability.
Cells were incubated under aerobic and two types of anaerobic atmosphere conditions, N2A or COA for 4 days, after which viability was measured via FACS analysis using PI penetration assay. Viability measurements (mean ± SD) from 3 independent experiments are shown.
Figure 3.
COA, but not N2A, results in S-phase arrest of cell cycle.
Cells were incubated under aerobic and anaerobic conditions, N2A or COA, for 4 days. Insert: representative FACS analysis of cell cycle for K562 cells incubated under different atmospheres. Arrows indicate center of S-phase. Main Figure: phase distribution of viable cells. Mean ± SD from 3 independent experiments is shown. Empty bars - G0/G1 phase, black bars - S phase and grey bars - G2/M phase.
Figure 4.
COA induces erythroid differentiation demonstrated by GPA expression and Hb synthesis.
Cells were incubated under aerobic and anaerobic conditions for 4 days. (A) Cell suspensions were centrifuged and pellets were photographed. Note the prominent red color in the CO-cell pellet. (B) FACS analysis of GPA expression. Dashed line: auto-florescence, black - air, blue - N2A and red - COA. Note the scale differences on the Y-axis. (C) Average cellular Hb content under different conditions using “Hb quantification assay” (see Material and Methods for details). Mean ± SD from 3 independent experiments are shown.
Figure 5.
COA induces morphological changes typical of different stages of erythroid maturation.
Cells were incubated for 4 days under air or COA. (A) Cytospin slides of cells stained with Giemsa-May-Grünwald. Arrows point to COA-cells in various maturation stages. Note the nuclei positioned toward the cell periphery and their condensation. Insert: terminal differentiation as seen by enucleation. (B) Cells were double stained with Hoechst 33342 (blue) and α-GPA (red) and then examined by fluorescence microscopy. Arrows indicate the positive GPA and negative Hoechst stained enucleated cells. (C) Cell morphology by Scanning Electron Microscopy (SEM). Air panel - a typical immature cell is shown, COA panel - cell size and concave shape typical for erythrocyte. (D) FACS analysis of GPA expression and cell size (sample of 10,000 cells). Square-enclosed parameters typical of differentiated of erythroid cells: small diameter due to condensation and population expressing high level of GPA.
Figure 6.
COA in enriched medium induces erythrocytes Hb synthesis.
Cells were incubated for 3 days in regular or enriched medium (see Material and Methods for details). Hb content of cytosols was measured using “Hb quantification assay”. Compositions of media included: (1) medium only; (2) medium supplemented with “heme required nutrients”; (3) highly enriched medium; RBC - Hb content of mature erythrocytes.
Figure 7.
1%COA suffices for cell viability preservation and elevated average Hb content.
Cells were incubated anaerobically with COA, N2A or 1% COA for 4 days in fully enriched media (details in Fig. 6, bar 3). Hatched bar - COA, black - 1% COA and grey - N2A. (A) Cell viability as assessed by TB assay. Note discontinuous Y-axis. (B) Average Hb content in cell population as measured by “Hb quantification assay” (see Material and Methods for details).
Figure 8.
1% COA-cells contain a subpopulation with elevated Hb content.
Cells were grown for 4 days in highly enriched medium under 1% COA and sorted by FACS for higher (H-GPA) and lower (L-GPA) GPA expression. (A) Grey area: GPA of total cell population. Black area: cells with H-GPA population. White area: cells with L-GPA population. (B) Hb content of sorted cells was measured by “Hb quantification assay” (see Material and Methods for details). White bar: L-GPA average Hb level. Black bar: H-GPA average Hb level.
Figure 9.
Schematic presentation of CO production and function in the erythroblastic island.
Shaded Red - Hb at varying concentrations; Blue – nucleus; Green – CO. Stages of differentiation in EI erythroblasts are shown in a clockwise fashion. 1: Clearance of FLVCR-associated “free” heme by hemopexin. 2: HO-1 induction by FLVCR associated “free” heme. 3: Enucleation of terminally differentiated erythroblast in EI. 3a: Engulfment of nucleus containing Hb remnants. 3b: Reticulocyte movement toward blood circulation. 4: HO-1 induction by Hb heme remnants. 4a. Dispersion of Hb in central macrophage. 4b. Formation of “free” heme from Hb phagocytosed with nucleus. 4c: Induction of HO-1 by “free” heme. (The membrane attached HO-1 is shown in the macrophage center for illustration purpose only.) 5: CO production by HO-1, leading to terminal differentiation. 5a. CO dispersion reaching erythroblast. 5b. Nuclear condensation and peripheral shift. 5c. Accelerated Hb synthesis, followed by membrane-associated “free” heme. 5d. Terminal erythroid differentiation upon attaining maximal Hb content.