Fig 1: IL-8 promotes IPF MPC self-renewal. A: IPF and control MPC (n = 4 cell lines each) CXCR1 mRNA expression levels were quantified by Q-PCR. B and C: to analyze the effect of IL-8 on IPF and control MPC self-renewal, 5,000 cells in a single cell suspension were seeded per well in 24-well dishes containing methylcellulose and recombinant IL-8 as indicated. The cells were cultured for 7 days. B: colony number per 5,000 cells was quantified microscopically (n = 3). C: graph depicting colony size and phase contrast image of IPF colony formation. Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.
Fig 2: IPF MPCs colocalize with macrophages in the peripheral region of the fibroblastic focus. Immunohistochemical (IHC) was performed on human IPF lung tissue (n = 10 IPF patient specimens). A: shown are 2 fibroblastic foci. Hematoxylin and eosin (H&E) staining was used to identify the fibroblastic focus and IHC was performed using CD163 (macrophage marker) and SSEA4 and S100A4 (MPC markers) antibodies to assess distribution of macrophages and IPF MPCs in the focus. Asterisk denotes focus core. Bar = 50 µm (left) and 20 µm (middle and right). B: shown are serial 4-µm sections stained for H&E, procollagen, SSEA4, S100A4, and CD163. Shown are two fibroblastic foci. Procollagen expression was used to delineate the myofibroblast-rich focus core. IHC analysis of the foci demonstrated S100A4- and SSEA4-positive cells at the periphery of the focus. CD163-positive cells codistributed with SSEA4- and S100A4-expressing cells at the focus perimeter. Asterisk denotes focus core. Fibroblastic Focus 1: bar = 50 µm (top and middle) and 20 µm (bottom); Fibroblastic Focus 2: bar = 50 µm (top) and 20 µm (bottom). C: IHC analysis of IPF lung tissue for CXCR1 and CXCR2 expression and CD163. Left: dual staining for CXCR1 (brown) and CD163 (red). Shown in the inset is a CD163-positive macrophage (red) with CXCR1 labeling (brown) on the perimeter of the cell. Right: dual staining for CXCR2 (brown) and CD163 (red). Bar = 100 µm (top) and 50 µm (bottom). Asterisk denotes focus core. D: in situ hybridization for IL-8 was performed on IPF lung tissue. Bar = 50 µm (top and bottom left) and 20 µm (bottom right). E: H&E staining and IHC was performed on human control lung tissue (n = 3 control patient specimens) using antibodies to S100A4, SSEA4, and CD163. Bar = 50 µm (H&E) and 20 µm (IHC).
Fig 3: Protein expression of CXCR1, CXCR2 and IL-8 in liver biopsy specimens of liver cancer (magnification, ×400). Protein expression of CXCR1 in (A) normal controls and (B) patients with liver cancer. (C) Relative mean density analysis showed differences in hepatic CXCR1 staining between liver cancer (n=30) and control tissues (n=12). Protein expression of CXCR2 in (D) normal controlsand (E) patients with liver cancer. (F) Relative mean density analysis showed the difference in hepatic CXCR2 staining between liver cancer (n=30) and controls (n=12). Protein expression of IL-8 in (G) normal controls and (H) patients with liver cancer. (I) Relative mean density analysis showed the difference in hepatic IL-8 staining between liver cancer (n=30) and control tissues (n=12). All data are presented as the mean ± SD and were analyzed with the two-tailed unpaired t-test (?P<0.05). CXCR, CXC chemokine receptor; IL-8, interleukin-8.
Fig 4: IL-8 promotes human liver cancer cell invasion and metastasis through CXCR1/2 receptors. (A) Huh7 and HepG2 cells were treated with IL-8 for 24 h, and the expression levels of CXCR1 and CXCR2 were detected by reverse transcription-quantitative polymerase chain reaction analysis and western blotting. (B) Huh7 cell migration 24 and 48 h after IL-8 stimulation was assessed using wound healing assays. (C) HepG2 cells migration after 24 and 48 h after IL-8 stimulation was assessed using wound healing assays. Right panel shows quantitation of the wound closure shown in the left panel. The y-axis represents the percentage of wound closure at 24 or 48 h post-wound introduction, as determined using ImageJ software. (D) Transwell assay analysis of the migration and invasion abilities of Huh7 and HepG2 cells (magnification, ×100). (E) Huh7 and HepG2 cells were pretreated with CXCR1 or CXCR2 antibodies (5 µM) for 30 min followed by stimulation with 10 ng/ml IL-8 for 24 h, and in vitro migration was measured with the wound healing assay. Results are expressed as the mean ± SEM; *P<0.05, compared with the control; #P<0.05, compared with the IL-8-treated group. CXCR, CXC chemokine receptor; IL-8, interleukin-8.
Fig 5: IL-8 promotes the proliferation and motility of IPF MPC progeny. A: CXCR1 mRNA expression levels were quantified in IPF and control MPC progeny (n = 4 cell lines each) by Q-PCR. B: shown is proliferation of IPF and control MPC progeny in response to recombinant IL-8. Cell number was quantified using the MTT Proliferation Assay Kit (n = 3). OD, optical density. C: shown is the migration of IPF and control MPC progeny in response to recombinant IL-8. Migration was quantified using the QCM Chemotaxis Cell Migration Assay Kit (n = 3). Data are expressed as means ± SE. P values were determined by two-tailed Student’s t-test.
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