Mediators of Inflammation / 2017 / Article / Fig 7

Research Article

Ubiquitin-Specific Protease 2 Modulates the Lipopolysaccharide-Elicited Expression of Proinflammatory Cytokines in Macrophage-like HL-60 Cells

Figure 7

USP2 may regulate cytokine expression by polyubiquitination of Oct transcription factors in LPS-stimulated HL-60 cells. (a) DNA binding activity determined by bead array. Differentiated USP2KD and mock-transfected control (Ctrl) HL-60 cells were treated with LPS (2.5 μg/mL) or vehicle alone for 1 h, and the nuclear fraction was subjected to analysis. A heatmap represents data from 29 transcription factors whose DNA binding activity increased during macrophage-like differentiation. NF-κB and Oct data are also shown as enlarged pictures. (b) A heatmap showing an abundance of transcripts for POU family transcription factors in mouse macrophages. (c) Detection of Oct family proteins in USP2KD and control cells following LPS stimulation. Signal intensities of the bands were measured and normalized to lamin-A levels. Data are presented as means ± SD of five samples. Representative images are also shown. versus control cells § versus 0 h. (d) Schematic summary of Oct-1/2 binding sites in the 5-proximal regions of the cytokine genes regulated by USP2. 5-proximal Oct-1/2 binding sites were predicted by presence of the octamer motif, ATTGCAT, and Oct-2 ChIP signals (ENCODE database) and also surveyed using public prediction tools (JASPAR and DECODE databases). Ten cytokine genes tightly controlled by USP2 were analyzed. (e) The DNA binding ratio of Oct-1 to Oct-2 at the TNF, CXCL8, IL6, and CCL4 promoters. ChIP-PCR was performed using five replicates. Data were normalized with those of input samples and represented as box-and-whisker plots. Oct-binding regions used for ChIP assays are depicted on each graph. (f) Detection of total K48- and K63-linked polyubiquitination on Oct proteins. Halo-tagged Oct-1 or Oct-2 and V5-tagged USP2A or mock plasmids with a HA-ubiquitin plasmid were introduced into HEK293FT cells for two days. Halo-tagged Oct proteins were pulled down and subjected to Western blot analysis. Input samples were also loaded as a reference. The HA-tag, K48- and K63-linked polyubiquitination chains (K48Ub and K63Ub), Oct-1, Oct-2, and the V5-tag were detected. Representative blot images are shown.
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