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1.
Figure 1

Figure 1. Identification of YS-HSCs.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

(a, b, and c) Immunohistochemistry staining of CD41 in E9 murine embyo yolk sacs. The arrows refer to cells expressing CD41 in E9 murine embryo yolk sacs. Magnifications: (a) 200×; (b) 400×; and (c) A representative section of H & E staining of the E9 murine embryo yolk sacs (magnification of 200×).

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
2.
Figure 8

Figure 8. Effects of HQ on Cyp4F18 protein expression.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Immunoblot analysis of Cyp4f18 proteins on HQ-treated YS-HSC (a) and BM-HSC (b) cells. Immunoblotting of Cyp4F18 was shown in the upper panels. GAPDH was measured as loading control. Densitometry of immunoblot results was shown in the lower panels. Values are presented as means ± SDs (n = 3–5). *, P<0.05; **P<0.01; compared with controls.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
3.
Figure 10

Figure 10. DSB repair induced by HQ in BM-HSC.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Representative microscopic fluorescence images of γ-H2AX foci accumulation after HQ treatment for 6 h. Nuclei were stained using PI and Al-exa488-labeled secondary antibodies, and were examined by confocal microscopy (oil, 200×). The experiments were repeated for three more times. Green fluorescence indicates formation of γ-H2AX foci.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
4.
Figure 2

Figure 2. Immunophenotyping of CD117+ yolk sac cells.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Representative flow cytometric data from more than three independent analyses were shown. The cells were fluorescently stained with CD117-PE. To show the CD117+ cell purity, CD117+ yolk sac cells before separation were shown on the left panel; CD117+ yolk sac cells after separation by using the anti-murine CD117 antibodies conjugated to mini-magnetic beads were shown on the right panel.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
5.
Figure 7

Figure 7. Effect of HQ on Cyp4f18 mRNA expression.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Expression of Cyp4f18 mRNA in YS-HSC (a) and BM-HSC (b) was measured by quantitative real–time PCR. The relative expression of target genes was calculated using the 2−ΔΔCt method. Data represent means ± SDs of at least three individual experiments (*, P<0.05; **, P<0.01; and #, P<0.001; compared to control group values.).

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
6.
Figure 9

Figure 9. Effect of hydroquinone on DNA-PKcs mRNA expression.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

YS-HSC (a) and BM-HSC (b) cells were treated with HQ at increasing concentrations and expression of DNA-PKcs mRNA was measured by quantitative real–time PCR. The relative expression of target genes was calculated using the 2−ΔΔCt method. Data represent means ± SDs of at least three individual experiments (*, P<0.05; **, P<0.01; and #, P<0.001; compared to control group values.).

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
7.
Figure 6

Figure 6. Induction of p53 by HQ.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

YS-HSC (a) and BM-HSC (b) were treated with increasing concentrations of HQ for 24 h. Total cell lysates were analyzed by immunoblotting with antibodies against p53. Expression of the GAPDH protein was used as a loading control. Upper panels: representative blotting results. Densitometry of the immunoblotting results was shown in the lower panels. Values are presented as means ± SDs (n = 3–5). * P<0.05.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
8.
Figure 4

Figure 4. Comparison of cytotoxicities of HQ on BM-HSC and YS-HSC.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Cells were exposed to increasing concentrations of HQ after 24 h liquid culture. (a) Cell viability was measured by the trypan blue exclusion assay. The initial cell amount used was 1×105/ml. Relative cell viability was represented as the percentage of viability of treated group over the control. (b) Cell viability was determined by the CCK-8 assay. Left: Viability of BM-HSC and YS-HSC; right: Relative viability of BM-HSC and YS-HSC over control. Data represent means ± SDs (n = 3).

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
9.
Figure 5

Figure 5. Comparison of apoptotic effects of HQ on BM-HSC and YS-HSC.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Cells were exposed to various concentrations of HQ after 24 h liquid culture and were stained with Annexin V and PI. Apoptosis was determined using flow cytometry as described in Materials and Methods. Illustrated is a representative of three separate experiments. (a) Percent of total apoptotic cells of YS-HSC. (b) Percent of total apoptotic cells of BM-HSC. (**P<0.01, compared to control group values). (c) A representative flow cytometry data showing apoptotic effects of HQ on BM-HSC and YS-HSC at the 5 µM concentration.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.
10.
Figure 3

Figure 3. Effect of HQ on CFU of YS-HSC and BM-HSC after 7 days of culture in vitro.. From: Comparison of Toxicity of Benzene Metabolite Hydroquinone in Hematopoietic Stem Cells Derived from Murine Embryonic Yolk Sac and Adult Bone Marrow.

Five thousand cells were sorted directly into the methylcellulose medium containing 0, 1.25, 2.5, or 5.0 µM hydroquinone supplemented with SCF, IL-3, IL-6, and Epo. Colony numbers were counted on day 7. Each experiment was done in triplicates. Quantitative data represents the average number of colonies per dish. (a) Morphology of CFU-E/BFU-E of BM-HSC and YS-HSC after 7d culture. (b) Morphology of CFU-GM of BM-HSC and YS-HSC after 7d culture. (c) CFU numbers of BM-HSC and YS-HSC. (d) Comparison of total CFU as percent of control between BM-HSC and YS-HSC. (e) Comparison of total CFU as percent of control between BM-HSC and YS-HSC treated with HQ at 1.25 µM. Data represent means ± SDs of three or more separate experiments (*, P<0.05; **, P<0.01; and #, P<0.001). CFU-GM includes CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), and CFU-granulocyte macrophage (CFU-GM). CFU-E represents colony-forming unit-erythroid. BFU-E represents burst-forming unit-erythroid.

Jie Zhu, et al. PLoS One. 2013;8(8):e71153.

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