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Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability

Junting Cai, Jianxin Wei, Valerie Schrott, Jing Zhao, Grant Bullock, Yutong Zhao
DOI: 10.1136/jim-2017-000622 Published 3 November 2017
Junting Cai
1Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
2Medical School, Xiangya Hospital of Central South University, Changsha, Hunan, China
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Jianxin Wei
1Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Valerie Schrott
3Department of Pathology, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Jing Zhao
1Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Grant Bullock
3Department of Pathology, Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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Yutong Zhao
1Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
4Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
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    Figure 1

    Ex vivo CD34+ human hematopoietic progenitor cell culture model of erythropoiesis. (A) Illustration of the morphological changes that occur during erythropoiesis. (B) Representative images of cells from days 1, 5, and 7 of erythroid differentiation. Cells were cytospun, fixed, and stained with the May-Grunwald and Giemsa stains. Bar, 15 nm. IL, interleukin; SCF, stem cell factor; EPO, erythropoietin.

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    Figure 2

    Ubiquitin-specific protease 50 (USP50) is increased during erythropoiesis. (A) Total RNA was extracted from human CD34+ hematopoietic progenitor cells (HPCs) on day 1 and day 3 of erythroid differentiation. Real-time quantitative PCR was used to compare ubiquitin-specific protease mRNA levels. Amplification of each sample was normalized to its glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript levels. Shown are the ratios of day 3/day 1 in a representative experiment from five independent experiments. (B) Total RNAs were extracted on days 1, 3, 5, and 9 from HPCs. USP50 mRNA levels were measured by real-time qPCR. Shown are data in a representative experiment from five independent experiments. (C) Whole cell lysates were made from HPCs on days 0, 1, 3, 5, and 9 of erythroid differentiation. USP50 and β-actin proteins were detected by immunoblot. Representative blots were shown from five independent experiments. 

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    Figure 3

    Ubiquitin-specific protease 50 (USP50) is localized in the nuclei and its binding partners are identified. (A) Mouse lung epithelial (MLE12) cells were transfected with USP50-V5 plasmids and incubated for 24 hours. Cells were fixed and stained with a V5 antibody. USP50-V5: green; DAPI (4',6'-diamidino-2-phenylindole, nuclear DNA stain): blue. (B) HEK293T cells were transfected with USP50-V5 plasmid and incubated for 24 hours. Immunoprecipitation with a V5 antibody, followed by SDS-PAGE and LC-MS/MS analysis. Shown are potential USP50 binding partners separated by known intracellular location or function based on literature.

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    Figure 4

    Ubiquitin-specific protease 50 (USP50) reduces Ku70 protein stability. (A) Mouse lung epithelial (MLE12) cells were transfected with 0–4 µg USP50-V5 plasmids per 35 mm dish and incubated for 24 hours. Total RNA was extracted and Ku70 mRNA levels were compared by real-time qPCR. The expression of Ku70 transcripts was normalized to Gapdh. The results were also normalized to 0 µg of USP50 plasmid. (B) MLE12 cells were transfected with USP50-V5 plasmids (0–4 µg of DNA per 35 mm dish) and incubated for 24 hours. Protein expression in whole cell lysates was analyzed by immunoblotting with antibodies against Ku70, V5, and β-actin. (C) MLE12 cells were transfected with USP50-V5 plasmid and incubated for 24 hours. Cells were treated with cycloheximide (CHX) (40 µg/mL) for 0–8 hours. Expression of proteins in whole cell lysates was analyzed by immunoblotting with antibodies against Ku70, V5, and β-actin.

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    Figure 5

    Ubiquitin-specific protease 50 (USP50) is a not stable protein. (A) Mouse lung epithelial (MLE12) cells were transfected with USP50-V5 plasmids and incubated for 24 hours. Cells were treated with cycloheximide (CHX) (40 µg/mL) with or without MG132 (20 µg/mL) or leupeptin (20 µg/mL) for 0–2 hours. Cell lysates were analyzed by immunoblotting with antibodies against V5 and β-actin. (B) MLE12 cells were transfected with USP50-V5 and treated with 5 µg/mL lipopolysaccharide (LPS) for 5 hours. Total RNA was extracted and USP50 mRNA levels were determined by real-time qPCR. The expression of USP50 transcripts was normalized to Gapdh.

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Journal of Investigative Medicine: 70 (8)
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Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability
Junting Cai, Jianxin Wei, Valerie Schrott, Jing Zhao, Grant Bullock, Yutong Zhao
Journal of Investigative Medicine Nov 2017, jim-2017-000622; DOI: 10.1136/jim-2017-000622

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Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability
Junting Cai, Jianxin Wei, Valerie Schrott, Jing Zhao, Grant Bullock, Yutong Zhao
Journal of Investigative Medicine Nov 2017, jim-2017-000622; DOI: 10.1136/jim-2017-000622
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Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability
Junting Cai, Jianxin Wei, Valerie Schrott, Jing Zhao, Grant Bullock, Yutong Zhao
Journal of Investigative Medicine Nov 2017, jim-2017-000622; DOI: 10.1136/jim-2017-000622
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