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Promotion of Erythropoietic Differentiation in Hematopoietic Stem Cells by SOCS3 Knock-Down

Liu, Yu-xiao, Dong, Xing, Gong, Feng, Su, Ning, Li, Su-bo, Zhang, Hai-tao, Liu, Jia-ling, Xue, Jing-hui, Ji, Shou-ping, Zhang, Zhi–wen·2015·PLOS ONE
DOI 10.1371/journal.pone.0135259PMC PMC4529111
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Fig 1 Identification of transfected HSCs. (A) GFP expression in HSCs was analyzed by Flow cytometric. (B) GFP expression in HSCs was analyzed by Immuno-fluorescence staining after sorting. Almost all of CD34+ cells had a strong GFP expression. Bar = 50 μm. Expression of SOCS3 in HSCs was examined by Q-PCR and western-blot after SOCS3 knock-down (C) or over-expression (D). The average of the normalized ratio of the target gene/GAPDH was calculated. The data represent the mean ± SEM from three experiments. ※comparing with shControl value<0.05, * comparing with Expression-control value<0.05
Fig 1 Identification of transfected HSCs. (A) GFP expression in HSCs was analyzed by Flow cytometric. (B) GFP expression in HSCs was analyzed by Immuno-fluorescence staining after sorting. Almost all of CD34+ cells had a strong GFP expression. Bar = 50 μm. Expression of SOCS3 in HSCs was examined by Q-PCR and western-blot after SOCS3 knock-down (C) or over-expression (D). The average of the normalized ratio of the target gene/GAPDH was calculated. The data represent the mean ± SEM from three experiments. ※comparing with shControl value<0.05, * comparing with Expression-control value<0.05CC BY · PubMed Central
Fig 2 SOCS3 regulated the fate of HSCs. (A) hematopoietic colonies from HSCs. 14 day after seed in methylcellulose, HSCs could form various types of hematopoietic colonies mainly including CFU-M, CFU-GM and BFU-E. Bar = 50 μm. (B) BFU-E and CFU-E derived from shSOCS3-1 CD34+ cells. Arrow directed CFU-E. Bar = 100 μm. (C) Quantification of erythroid colonies in different groups. The numbers of erythroid colonies increased by about 3-fold with SOCS3 down-expression and decreased by about 2.5-fold with SOCS3 overexpression. The data represent the mean ± SEM from three experiments.※comparing with shControl value<0.05, * comparing with Expression-control value<0.05. (D) Photographs of cytospin preparations of CFU-E from shSOCS3-1 CD34+ cells stained by DAPI. The arrows directed enucleated cells. Bar = 50 μm.
Fig 2 SOCS3 regulated the fate of HSCs. (A) hematopoietic colonies from HSCs. 14 day after seed in methylcellulose, HSCs could form various types of hematopoietic colonies mainly including CFU-M, CFU-GM and BFU-E. Bar = 50 μm. (B) BFU-E and CFU-E derived from shSOCS3-1 CD34+ cells. Arrow directed CFU-E. Bar = 100 μm. (C) Quantification of erythroid colonies in different groups. The numbers of erythroid colonies increased by about 3-fold with SOCS3 down-expression and decreased by about 2.5-fold with SOCS3 overexpression. The data represent the mean ± SEM from three experiments.※comparing with shControl value<0.05, * comparing with Expression-control value<0.05. (D) Photographs of cytospin preparations of CFU-E from shSOCS3-1 CD34+ cells stained by DAPI. The arrows directed enucleated cells. Bar = 50 μm.CC BY · PubMed Central
Fig 3 FACS analysis of cell surface markers. On day 7 after liquid culture, cells in different groups were harvested and stained with combinations of fluorescently labeled antibodies against markers characteristic for erythroid lineage.
Fig 3 FACS analysis of cell surface markers. On day 7 after liquid culture, cells in different groups were harvested and stained with combinations of fluorescently labeled antibodies against markers characteristic for erythroid lineage.CC BY · PubMed Central
Fig 4 Erythroid specific markers were analyzed by FACS. On day 14 and 21 in liquid culture, cells in different groups were stained with combinations of fluorescently labeled antibodies against markers characteristic for erythroid lineage.
Fig 4 Erythroid specific markers were analyzed by FACS. On day 14 and 21 in liquid culture, cells in different groups were stained with combinations of fluorescently labeled antibodies against markers characteristic for erythroid lineage.CC BY · PubMed Central
Fig 5 Characterization of erythroid cells from HSCs. (A) Photographs of cytospin preparations of the erythroid cells. Cells from shSOCS3-1 or shcontrol CD34+ cells were stained by Wright-Giemsa on day 21 in liquid culture. The arrows directed enucleated cells. Bar = 50um. (B) Statistical analysis for the percent of enucleated cells in different groups. Enucleated cells on day 21 in liquid culture; the percent of enucleated cells was calculated as the ratio enucleated cells / all cells in a field. Data represent mean percent of positive cells ±SEM. n = 5, *p < 0.05. (C) Functional assays of erythroid cells from shSOCS3-1 CD34+ cells. Oxygen dissociation curves of RBCs derived from shSOCS3-1 CD34+ cells or human adult blood.
Fig 5 Characterization of erythroid cells from HSCs. (A) Photographs of cytospin preparations of the erythroid cells. Cells from shSOCS3-1 or shcontrol CD34+ cells were stained by Wright-Giemsa on day 21 in liquid culture. The arrows directed enucleated cells. Bar = 50um. (B) Statistical analysis for the percent of enucleated cells in different groups. Enucleated cells on day 21 in liquid culture; the percent of enucleated cells was calculated as the ratio enucleated cells / all cells in a field. Data represent mean percent of positive cells ±SEM. n = 5, *p < 0.05. (C) Functional assays of erythroid cells from shSOCS3-1 CD34+ cells. Oxygen dissociation curves of RBCs derived from shSOCS3-1 CD34+ cells or human adult blood.CC BY · PubMed Central
Fig 6 The effect of SOCS3 expression on the gene expression profile of HSCs. Heat-map showing gene expression profiles in CD34+ cells 48 h after transduction with shSOCS3-1 or shcontrol vectors.
Fig 6 The effect of SOCS3 expression on the gene expression profile of HSCs. Heat-map showing gene expression profiles in CD34+ cells 48 h after transduction with shSOCS3-1 or shcontrol vectors.CC BY · PubMed Central
Fig 7 GO analysis of SOCS3-induced gene expression alteration in HSCs. Branches of the GO hierarchical tree with significantly enriched GO terms are indicated in red boxes, Insignificant GO terms within the hierarchical tree are shown as white boxes.
Fig 7 GO analysis of SOCS3-induced gene expression alteration in HSCs. Branches of the GO hierarchical tree with significantly enriched GO terms are indicated in red boxes, Insignificant GO terms within the hierarchical tree are shown as white boxes.CC BY · PubMed Central

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Citations

Cites

Research works printed but not linked: 32

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Metadata

Authors
Liu, Y.-X., Dong, X., Gong, F., Su, N., Li, S.-B., Zhang, H.-T., Liu, J.-L., Xue, J.-H., Ji, S.-P., & Zhang, Z.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-08-07
Volume
10
Issue
8
Pages
e0135259
ISSN
1932-6203
DOI
10.1371/journal.pone.0135259
PMC
PMC4529111
Cited by
5
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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