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Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

Zhang FL, Shen GM, Liu XL, Wang F, Zhao YZ, Zhang JW - J. Cell. Mol. Med. (2012)

Bottom Line: Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression.Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo.Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

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Flow cytometry analysis of CD235a-positive cells. (A) The K562 cells were untreated and unlabelled as a negative control. (B) The K562 cells were untreated but labelled with IgG isotype control. (C) The K562 cells transfected with siRNA control for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. (D) The K562 cells transfected with siRNA targeting HIF1A for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. M1 mark indicates the CD235a-positive peak. MFI, mean fluorescent intensity.
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fig07: Flow cytometry analysis of CD235a-positive cells. (A) The K562 cells were untreated and unlabelled as a negative control. (B) The K562 cells were untreated but labelled with IgG isotype control. (C) The K562 cells transfected with siRNA control for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. (D) The K562 cells transfected with siRNA targeting HIF1A for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. M1 mark indicates the CD235a-positive peak. MFI, mean fluorescent intensity.

Mentions: We also performed flow cytometry analysis to assay expressions of the erythroid surface markers CD71 and CD235a. As the curve shifted right, the expression of CD71 increased in K652 cells exposed to hypoxia for 32 hrs or 48 hrs (Fig. 6A), and significantly enhanced in K562 cells treated with DFO or CoCl2 (Fig. 6B). In addition, the expression of CD235a gradually enlarged under hypoxia (Fig. 6C), and also notably increased in DFO- or CoCl2-treated K562 cells (Fig. 6D). While CD71 (also known as transferrin receptor) has been verified as a target gene of not only HIF1 but also GATA1 [24-26], we additionally determined if the expression of CD235a was affected by siRNAs that targets HIF1A or GATA1. The expression of CD235a obviously decreased when the K562 cells with knocked down of HIF1A or GATA1 were cultured in normoxia (Fig. 6E). Under hypoxic conditions, a significantly reduced expression of CD235a in K562 cells with knockdown of GATA1 was detected, while a slightly decreased expression of CD235a in K562 cells with HIF1A knockdown (Fig. 6F). Furthermore, when a positive peak was marked according to the negative control, a notably decreased percentage of CD235a-positive cells was detected in the K562 cells with HIF1A knockdown (Fig. 7). These showed that knockdown of HIF1A or GATA1 leads to a decreased expression of CD235a.


Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

Zhang FL, Shen GM, Liu XL, Wang F, Zhao YZ, Zhang JW - J. Cell. Mol. Med. (2012)

Flow cytometry analysis of CD235a-positive cells. (A) The K562 cells were untreated and unlabelled as a negative control. (B) The K562 cells were untreated but labelled with IgG isotype control. (C) The K562 cells transfected with siRNA control for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. (D) The K562 cells transfected with siRNA targeting HIF1A for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. M1 mark indicates the CD235a-positive peak. MFI, mean fluorescent intensity.
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Related In: Results  -  Collection

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fig07: Flow cytometry analysis of CD235a-positive cells. (A) The K562 cells were untreated and unlabelled as a negative control. (B) The K562 cells were untreated but labelled with IgG isotype control. (C) The K562 cells transfected with siRNA control for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. (D) The K562 cells transfected with siRNA targeting HIF1A for 24 hrs and cultured under hypoxic conditions for another 24 hrs, and then labelled with PE-anti-CD235a. M1 mark indicates the CD235a-positive peak. MFI, mean fluorescent intensity.
Mentions: We also performed flow cytometry analysis to assay expressions of the erythroid surface markers CD71 and CD235a. As the curve shifted right, the expression of CD71 increased in K652 cells exposed to hypoxia for 32 hrs or 48 hrs (Fig. 6A), and significantly enhanced in K562 cells treated with DFO or CoCl2 (Fig. 6B). In addition, the expression of CD235a gradually enlarged under hypoxia (Fig. 6C), and also notably increased in DFO- or CoCl2-treated K562 cells (Fig. 6D). While CD71 (also known as transferrin receptor) has been verified as a target gene of not only HIF1 but also GATA1 [24-26], we additionally determined if the expression of CD235a was affected by siRNAs that targets HIF1A or GATA1. The expression of CD235a obviously decreased when the K562 cells with knocked down of HIF1A or GATA1 were cultured in normoxia (Fig. 6E). Under hypoxic conditions, a significantly reduced expression of CD235a in K562 cells with knockdown of GATA1 was detected, while a slightly decreased expression of CD235a in K562 cells with HIF1A knockdown (Fig. 6F). Furthermore, when a positive peak was marked according to the negative control, a notably decreased percentage of CD235a-positive cells was detected in the K562 cells with HIF1A knockdown (Fig. 7). These showed that knockdown of HIF1A or GATA1 leads to a decreased expression of CD235a.

Bottom Line: Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression.Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo.Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.

Show MeSH
Related in: MedlinePlus