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. 2018 Dec 21;9:5426. doi: 10.1038/s41467-018-07126-9

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — PHD2 expression and function in human melanocytes and melanomas. a IHC staining of PHD2 in 126 nevus and 266 melanoma specimens. A melanoma progression tissue microarray (TMA) was stained with the anti-PHD2 antibody and PHD2 staining positive and negative cases were calculated. Representative PHD2 positive (upper panel) and negative cases (lower panel) are shown. Bars indicate 100 µm. b Percentage of cases with PHD2 expression. There was significantly less percentage of melanoma cases with PHD2 expression than that of nevi; *p < 0.01. c. TCGA SKCM patients were sorted into PHD2-high and PHD2-low groups according to PHD2 mRNA expression levels (top 5% versus bottom 5%). d PHD2 expression and survival curve. The Kaplan–Meier survival curves showed that the PHD2-high group have a significantly improved patient survival than PHD2-low group (log rank p value = 0.00643). e PHD2 knockdown induces Akt phosphorylation. Immortalized human melanocytes (hTERT/p53DD/Cdc24R24C-BRAFV600E) were transfected with two independent PHD2 shRNAs. Cells transfected with shGFP were included as a negative control. Western blots were performed using the indicated antibodies. f Reintroduction of PHD2 in PHD2^−/− MEFs reduces Akt phosphorylation. PHD2^−/− MEFs (left lane) were transfected with wild-type PHD2 (lane adjacent to the left lane). PHD2-positive MEFs which express endogenous PHD2 were used as a control (right lane). g DFO or DMOG treatment. Immortalized human melanocytes were transfected with control vectors or EGLN knockdown vectors. These cells were than treated with DFO or DMOG. h Overexpressed HIF-2α in immortalized human melanocytes. i The melanoma-derived PHD2-P317 mutation inhibits PHD2 binding to HIF-1α. Biotin-labeled HIF1α-ODD peptides were incubated with WCLs derived from HEK293T cells transfected with the indicated PHD2 constructs (beads as negative control). Binding of PHD2 to HIF1α-ODD is abolished with the P317 mutation. j The PHD2-P317 mutation inhibits PHD2 and HIF-1α interaction. Unlike WT-PHD2, P317S-PHD2 is largely impaired in inhibiting HRE reporter activities detected by the Dual-Luciferase Reporter Assay System. The error bars indicate s.d.