Research Article

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

Figure 2

USP2 knockdown (KD) causes an increase in the expression of proinflammatory cytokines. USP2KD (KD) and mock-transfected control (Ctrl) cells were treated with PMA (30 nM; 1 d) and subsequently stimulated with LPS (2.5 μg/mL; a–e, h), Pam3CSK4 (5 μg/mL; f, g), or vehicle alone for (h) 1 h, (a–d, f) 4 h, or (e, g) 12 h. mRNA levels of (a) USP2 splice variants and (d, f) cytokines were determined by qRT-PCR and normalized according to the level of HPRT-1 mRNA. (b) Western blot analysis of USP2 protein. Signal intensities of the bands were measured and normalized to the level of GAPDH. Representative images are shown. (c) Schematic summary of the comprehensive cytokine expression analysis. mRNA levels of 104 cytokines were determined by qRT-PCR. Triplicate samples were used in the analysis. (e, g) The IL-6 concentration in the supernatant of USP2KD cells was determined by ELISA. (h) Chromatin accessibility of 5 flanking region of IL6, TNF, and CXCL8 in USP2KD cells. The RPS6 gene was used as a positive reference. Data are presented as means ± SD of (a, d, f) six, (b) three, (e, g) eight, or (h) five samples. versus control cells; § versus vehicle-treated cells.