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. 2017 Dec 13;8:2116. doi: 10.1038/s41467-017-02029-7

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© The Author(s) 2017

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 — An in vivo genetic screen identifies OTUD1 as potent suppressor of breast cancer metastasis. a, b Flow chart and figures of the in vivo screen identifying DUBs that inhibit breast cancer metastasis. Low metastatic MDA-MB-231-Luciferase/GFP breast cancer cells were infected with lentiviruses expressing DUB shRNAs and intracardially injected into nude mice. The mice were monitored for 4 weeks by in vivo bioluminescent imaging (BLI) and the early metastatic nodules were isolated and the corresponding shRNAs were identified by sequencing. See Supplementary Fig. 1a for details. c Immunoblot (IB) analysis of OTUD1 shRNA and sgRNA-mediated knockdown and knockout in MDA-MB-231 (BM) cells. d IB analysis of MDA-MB-231 (BM) cells stably expressing doxycycline (dox)-inducible OTUD1 and treated with the indicated doses of Dox. e Left panel: BLI of three representative mice injected with MDA-MB-231 control cells or cells deficient in OTUD1, Images were taken 4 weeks after injection. Two independent sgRNAs were used to generate OTUD1 knockout cells. Right upper panel: BLI signals of all mice in each experimental group at week 5. Right lower panel: The percentage of metastasis-free mice in each experimental group followed in time. f MDA-MB-231 cells expressing DOX-inducible OTUD1-wt/CA was intracardially injected in mice. DOX was administrated 21 days after inoculation of the cells. Metastasis was analyzed by BLI. Left panel: BLI of representative mice from each group at indicated days. Right upper panel: BLI signal of each experimental group followed in time. Right lower panel: The percentage of metastasis-free mice in each experimental group followed in time. g Oncomine database summary for OTUD1 expression in multiple cancers (left). Box plots of OTUD1 expression levels in invasive ductal breast carcinoma (IDBC) and lung adenocarcinoma (LA) compared with normal tissue (right). h Kaplan–Meier analysis of relapse-free survival of patients in publicly available breast cancer datasets (NKI 295). i BRCA1 target genes are enriched in OTUD1-high versus OTUD1-low expressing patients shown by pre-ranked gene-set enrichment analysis (GSEA). *p < 0.05, **p < 0.01 and ***p < 0.001 (two-tailed Student′s t-test (e, f), Log-rank test (h) or two-way analysis of variance (ANOVA) (e, f))