Journal: Scientific Reports
Article Title: Global functional profiling of human ubiquitome identifies E3 ubiquitin ligase DCST1 as a novel negative regulator of Type-I interferon signaling
doi: 10.1038/srep36179
Figure Lengend Snippet: ( A ) IFNβ treatment modulates transcription of several human ubiquitome genes negatively regulating of IFN-I signaling. HEK293T cells were treated with human 500 units/ml IFNβ for 4, 12 and 24 hours, and gene expression was quantified by q-RTPCR. The displayed values correspond to mean ± SD from a representative experiment executed in triplicates. The given values are presented as fold change in mRNA levels relative to unstimulated sample, taken as 1. The q-RTPCR results were determined by assessing relative Ct value, using the formula Fold-change = 2 (Ct of unstimulated – Ct of stimulated) Target gene /2 (Ct of unstimulated – Ct of stimulated) Reference gene . We compared values for untreated samples with each one of the interferon treated samples. ( B ) Endogenous DCST1 expression was upregulated in HEK293T cells at the protein level after IFNβ treatment. ( C ) DCST1 overexpression attenuated IFNβ induced enhancement of ISRE-luciferase reporter activity in a dose dependent manner, HEK293T cells. The displayed values are relative ISRE-luciferase activity normalized with the internal control renila luciferase values, and correspond to mean ± SD from a representative experiment. ( D ) Overexpression of 100 ng of DCST1 attenuated IFNβ induced enhancement of ISRE-GFP reporter activity in HEK293T cells. A representative fluorescence microscopy image (4x magnification) is shown. ( E ) Silencing of DCST1 using siRNAs enhanced ISRE-luciferase reporter activity driven by IFNβ stimulation in HEK293T cells. The displayed values are relative ISRE-luciferase activity normalized with the internal control renila luciferase values, and correspond to mean ± SD from a representative experiment. ( F ) siRNAs targeting DCST1 efficiently knocked down DCST1 protein expression. ( G ) siRNA mediated knockdown of DCST1 enhanced the expression of ISG RIG-I upon stimulation of HEK293T cells with IFNβ for 12 hrs. Si, siRNA; NT, non-targeting negative control siRNA; hrs, hours; EV, empty vector; ng, nanogram. Significance was calculated by using ANOVA test, where p < 0.05 was interpreted as significant. * p < 0.05; ** p < 0.01. Panels shown in Fig. 4(B,F,G) are cropped sections of full images. shows uncropped images corresponding to panels shown in Fig. 4(B,F,G).
Article Snippet: Recombinant human Interferon beta (IFNβ) 1b, expressed in E.coli was purchased from pbl assay science (#11420-1).
Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Luciferase, Activity Assay, Control, Fluorescence, Microscopy, Knockdown, Negative Control, Plasmid Preparation
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