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. 2018 Oct 26;9:4456. doi: 10.1038/s41467-018-06893-9

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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. 3 — P4HA1 enhances HIF-1α protein stability. a, b Analysis of HIF-1α protein degradation in control and shP4HA1-expressing MDA-MB-231 cells with cycloheximide (CHX, 100 μM) treatment (0, 5, 10, 20, 40, and 60 min). Results are presented as mean ± SEM. n = 3; *p < 0.05, independent Student’s t test. c, d Analysis of HIF-1α protein degradation in control and shP4HA1-transfected 293FT cells with cycloheximide (CHX, 100 μM) treatment (0, 5, 10, 20, 40, and 60 min). e Immunoprecipitation and western blot analysis of HIF-1α ubiquitination in 293FT cells transfected with HIF-1α-Flag and UB-HA construct, plus shCtrl or shP4HA1 plasmids. f Western blot assessing HIF-1α protein levels in control and P4HA1-silenced MDA-MB-231 cells treated with proteasome inhibitor Bortezomib (BTZ) for 12 h. g Analysis of hydroxylated HIF-1α (P402) levels in control and P4HA1-silenced MDA-MB-231 cells, control or P4HA1-expressing MCF10A cells. h, i ODD-luciferase reporter activity in control, P4HA1-expressing MCF10A cells, and P4HA1-silenced MDA-MB-231 cells under normoxia and hypoxia conditions. Results are presented as mean ± SEM; n = 3; **p < 0.01, *p < 0.05, one-way ANOVA test. j HIF-1α protein levels were assessed in control and P4HA1-silenced MDA-MB-231 cells with wild-type HIF-1α or proline hydroxylation-deficient HIF-1α PPAA overexpression.