Fig 1: The expression of S100A4 is up-regulated in C. rodentium-infected mouse colons. C57BL/6 mice were infected orally with C. rodentium as described in the Materials and Methods. Colons and other tissues were collected at different time points. (A) Real-time quantitative PCR analysis of S100A4 mRNA in various tissues from infected C57BL/6 mice on day 0 and day 7 after infection. GAPDH was used as the reference control, (n = 4); *P < 0.05, **P < 0.01. The mRNA level of liver of non-infected mice is set as 1.00 to calibrate the relative levels in other tissues. (B) Real-time quantitative PCR analysis of S100A4 mRNA in colons from infected WT mice on day 0, 7, 14, 21 after infection. GAPDH was used as the reference control, (n = 4); **P < 0.01. The mRNA level of non-infected mice (day 0) is set as 1.00 to calibrate the relative levels in other days. (C) Immunohistochemical staining for S100A4 (brown) in colon sections from C57BL/6 mice on day 0 (uninfected) and day 7, 14, and 21 after infection (n = 4). (D) The numbers of S100A4+ cells per HPF ( × 200), (n = 4); *P < 0.05, **P < 0.01. The experiment was performed by an observer blinded to the experimental condition. (E) Flow cytometry analysis of the phenotypes of S100A4+ cells in the colons of S100A4+/+.GFP mice after C. rodentium infection 7 days by staining GFP+ cells with CD45, F4/80, CD11c, CD4, CD8 and CD19 antibodies.
Fig 2: Infection-induced colitis and colonic pathology are attenuated in S100A4-deficient mice. Groups of WT and S100A4 −/− mice were infected orally with C. rodentium. (A) Representative colon figures from WT and S100A4 −/−mice (n = 3) on day 7 after infection. (B) Colon length from WT and S100A4 −/− mice (n = 3) on day 0 and day 7 after infection; *P < 0.05. (C) On day 0 (uninfected), day 7, 14, and 21 after infection, colons were sectioned and stained with H&E (n = 3). (D) Inflammation scores of WT mice and S100A4 −/− mice colon on day 0, 7, 14 and 21 p.i. The combined score equals the sum of the separate scores, including Edema in the sub-mucosa, PMN infiltration, reduced number of goblet cells and ulcerate epithelial layer, (n = 3); *P < 0.05, **P < 0.01. (E) Protein levels of some chemokines and inflammatory cytokines in the colons of WT and S100A4 −/− on day 7 p.i. with C. rodentium (n = 4), *P < 0.05, **P < 0.01.
Fig 3: S100A4 inhibited autophagy to promote tumor cell survival through ß-catenin signaling.a A549 cells were treated with HBSS and S100A4 as indicated. The indicated signal pathways were analyzed by western blot in A549 cells. b Western blot analysis of ß-catenin, phospho-ß-catenin, Bax, LC3-I/II and GAPDH protein levels in tumors from S100A4-/- and WT mice. A549 (c) and LLC (f) cells were transfected with plasmid overexpressing ß-catenin (pcDNA3.1-ß-catenin) or control plasmid (pcDNA3.1) and then treated with 1 µg/ml S100A4 in HBSS for 4 h. At 48 h after transfection, cell lysates were subjected to western blot analysis with the indicated antibodies. Cell viability of A549 cells (d) and LLC cells (g) treated with pcDNA3.1-ß-catenin was measured using a CCK-8 kit. mRNA levels of target genes (CyclinD1 and c-myc) in ß-catenin pathway from A549 (e) and LLC (h) cells was measured by RT-PCR; *p < 0.05, **p < 0.01
Fig 4: S100A4-induced autophagy in A549 cells is RAGE dependent.A549 and LLC cells were incubated with the RAGE-specific blocker FPS-ZM1 (75 nmol/µl) for 30 min before being treated with S100A4 (1 µg/ml) in HBSS for 4 h. The treated A549 cells (a) and LLC cells (c) were collected and analyzed for the expression of ß-catenin, phospho-ß-catenin, RAGE, Bax and LC3-I/II by western blot. Cell viability of A549 (b) and LLC (d) cells treated with FPS-ZM1 was determined by a CCK-8 kit. e A549 cells were transfected with GFP-LC3-I/II plasmid (1 ng/µl) for 6 h. f Representative images of GFP-LC3 staining in cells and quantification of LC3 puncta numbers per cell are shown; *p < 0.05, **p < 0.01
Fig 5: Upregulation of S100A4 expression in NSCLC tissues.Tissue microarray from 120 lung cancer patients was stained with anti-S100A4. a Representative S100A4 staining is shown at ×200 (upper) and ×400 magnifications (lower). Scale bar, 50 µm. b Percent of the cases expressing S100A4 in carcinoma tissues. c Percent of S100A4 negative, low and high expression in different tumor grades. d Average tumor size in NSCLC patients with different S100A4 protein levels
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