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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Hypoxia-induced Slug SUMOylation enhances lung cancer metastasis

Fig. 4

SUMOylation enhances the transcriptional repression activity of Slug. a Distribution of SUMOylated Slug in the cytosolic and chromatin-bound fractions. Cytosolic HSP90 protein and chromatin-bound Lamin B were used as controls for cellular fractionation. b The interaction of Slug with Ubc9 in the cytosolic and chromatin-bound fractions. HEK293T cells co-expressing 3xFlag-labelled Slug and HA–Ubc9 were harvested and immunoprecipitated using anti-Flag antibodies. HSP90 and Lamin B are shown as fractionation controls. c, d SUMOylation enhances the transcriptional repression activity of Slug. HEK293T cells were cotransfected with the indicated plasmids and a reporter vector driven by SBS–Gal4 (c) or by the E-cadherin promoter (d). Luciferase activities and immunoblotting were evaluated 36 h after transfection. The activity induced by Gal4–VP16 alone (c) or basal activity (d) was normalized to 100%. All data were reported as mean values ± SEM, and P values were calculated via Student’s t-test. The asterisk represents a P value of < 0.05 compared to the group stimulated with Gal4–VP16 alone or the basal activity level. e SUMOylation increases the recruitment of HDAC1. Lysates of HEK293T cells cotransfected with the indicated plasmids were subjected to immunoprecipitation using an anti-HA antibody. f Top: The indicated antibodies were used to pull down protein-DNA complexes, and the E-cadherin promoter level in the samples was determined by PCR using a gene-specific primer set. Input, an aliquot of each sample was prepared and used as a template for PCR to examine the level of the E-cadherin promoter before immunoprecipitation (IP). Bottom: Western blot analysis of the indicated proteins was performed on the products of ChIP. g SUMOylation affects the expression of Slug-regulated downstream targets. HEK293 and CL1–2 cells were induced to express the wild-type and mutant Slug, respectively, using a lentiviral system. The mRNA expression of the indicated genes was determined via RT-PCR. Gβ-like was used as the internal control

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