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. 2016 Dec 9;7:13644. doi: 10.1038/ncomms13644

Figure 1. HAUSP stabilizes HIF-1α to modulate its activity and knockdown of HAUSP reverses EMT and decreases metastatic activity.

Figure 1

(a) Interaction between HAUSP and HIF-1α using co-immunoprecipitation assays. (b) In vitro deubiquitination assays showed that HAUSP significantly decreased the levels of polyubiquitinated HIF-1α. The densitometer reading of polyubiquitinated HIF-1α levels was listed underneath each lane. The first lane was arbitrarily set as 1. (c) Stably expressed HAUSP in H1299 cells significantly increased the half-life of HIF-1α. Lower panel: the degradation curve by densitometric measurement to determine the half-life of endogenous HIF-1α proteins. (HAUSP-CS mutant: negative control). (d) The HIF-1αS366A mutant did not interact with HAUSP in co-immunoprecipitation experiments in 293T cells. (e) The polyubiquitinated HIF-1αS366A mutant could not be deubiquitinated by HAUSP. (f) Western blot analysis showed the increased HIF-1α levels through co-expression of wild-type HAUSP in 293T cells. β-actin, loading control; N, normoxia; H, hypoxia. (g) Further activation of the Twist1 promoter-driven reporter activity by co-expressing HIF-1α and HAUSP in transient transfection assays. The samples of transfecting with reporter alone (normoxia or hypoxia) were used as the controls. (h) Knockdown of HAUSP reversed EMT marker gene expressions and decreased the expression of HIF-1α downstream targets in H1299 cells that constitutively expressed HIF-1α. (i) Immunofluorescence staining showed the reversion of EMT marker gene expression in H1299 cells with HAUSP knockdown. Green fluorescence: E-cadherin, red fluorescence: vimentin. (j) Knockdown of HAUSP decreased the in vitro migration and invasion activity of H1299 cells. The H1299 scrambled-siRNA control clone was used as the control. (k) Representative photos of gross anatomy and histology pictures of H1299 cells (vector control, scrambled siRNA control versus knockdown of HAUSP) injected (tail vein or orthotopic) into nude mice. White arrows indicated tumour nodules. (l) The number of metastatic lung tumour nodules of H1299 cells (vector control, scrambled siRNA control versus knockdown of HAUSP). The number of mice used for each group was 6. The scrambled siRNA control was used as the control lane. For (g,j), data from three independent experiments are expressed as mean±s.d. For (l), data are presented as the mean±s.d. (n=6 mice/group).The asterisk (*) indicated statistical significance (P<0.05) between experimental and control clones or transfections, t-test.