hPTTG1 overexpression upregulates the expression of CXCR2, IL-8, and GROα. (A) Conditioned media were collected from MCF-7Mock or MCF-7hPTTG1 cells treated with vehicle control (DMSO) or an NF-κB inhibitor (JSH-23) for ELISA analysis of IL-8 (upper panel) and GROα (lower panel). Unconditioned DMEM media acted as negative control. (B) CXCR1 (upper panel) and CXCR2 (lower panel) mRNA expression was analyzed with qRT-PCR in MCF-7Mock and MCF-7hPTTG1 cells. (C) hPTTG1 and CXCR2 mRNA expression was analyzed with qRT-PCR in MCF-7Mock, MCF-7hPTTG1, MCF-10AMock, MCF-10AhPTTG1, and MDA-MB-231 cells. (D) MCF-10AMock, MCF-10AhPTTG1, MCF-7Mock, and MCF-7hPTTG1 cells were stained with a FITC-conjugated anti-CXCR2 antibody for FACS analysis. (E) The regulatory sequences of IL-8, GROα, and CXCR2 were co-transfected with pcDNA3.1 (Mock) or pcDNA3.1-hPTTG1 (hPTTG1) into MCF-7 cells for luciferase reporter assays. (F) Cell lysates were collected from untransfected MCF-7 (WT), MCF-7Mock, and MCF-7hPTTG1cells for immunoblot analysis. (G) The pGL4 vector (Mock), IL-8 promoter, or the mutated IL-8 promoter was transiently cotransfected with pcDNA3.1-hPTTG1 expression plasmid into MCF-7 cells for luciferase reporter assays. (H) The pGL4 vector (Mock), GROα promoter, or the NF-κB binding site deleted mutant was transiently cotransfected with the pcDNA3.1-hPTTG1 expression plasmid into MCF-7 cells for luciferase reporter assays. (I) ChIP assays were performed on MCF-7Mock or MCF-7hPTTG1 cells. The ChIP-qPCR data are expressed as the fold increase over the control (IgG) for the promoter region containing p65-binding elements. The results in A to C, E, and G to I are presented as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; NS, not significant.