Fig 1: Effect of AMF gene silencing on EC cells migration and invasionA. Ishikawa and HEC-1B cells were stably transfected with plasmid containing AMF-specific shRNA (shAMF-1 or shAMF-2)) or control plasmid (mock). Cells were then analyzed by qRT-PCR. B. Top, Immunoblot analysis for AMF protein and ß-actin expression; Bottom, quantification analysis of AMF expression. AMF cytokine itself was used as positive control. C. Intracellular PGI/AMF enzyme activity was measured in EC cell lines by enzymatic spectrophotometer assay. D. and E. Transwell assay for cells migration (D) and invasion (E) Cells were seeded onto the upper surface of Transwell chambers without (D) or with Matrigel-coated (E) After 16 h (D) or 24 h (E) of incubation, the penetrating cells were stained with calcein-AM and recorded under a microscope. Top, Photographs depict the migration or invasion of EC cells; Bottom, quantitative data are expressed as mean average ± SD from three independent experiments. (*, P < 0.05; **, P < 0.01, ***P < 0.001, compared with the control cells).
Fig 2: AMF/PGI mediates cell proliferation, migration and invasion activities through MAPK-ERK1/2 signalingA. and B. The effect of PGI on U0126-inhibited cells migration and invasion. Ishikawa/shAMF-1, HEC-1B/shAMF-1 cells and their mock controls were pretreated with U0126 or DMSO (control) for 24 hours, and cells that had migrated (left) or invaded (right) were counted after 16 h (left) or 24 h (right) in Transwell assays. Quantitative data from 3 different experiments are presented. C. The effect of PGI on U0126-inhibited cell proliferation. Cells treated as described above were then counted after 1 day, 3 days, and 5 days using MTS assays. Data represent the mean ± S.D. of the three independent experiments (**P < 0.01).
Fig 3: Autocrine motility factor is highly expressed in EC tissues and serumA. Immunohistochemical analysis of AMF and AMFR expression in normal endometrium and endometrioid cancer (EC). B. Summary of the IHC score in normal endometrium and EC (***p < 0.001). C. AMF expression in EC tissue specimens (n = 52) and normal endometrium (n = 30) was assessed by qRT-PCR and normalized to ß-actin expression (**P < 0.01, unpaired Student's t test). D. The levels of AMF in serum of patients with EC (n = 15) and normal endometrium (n = 15) were measured by ELISA (**P < 0.01, unpaired Student's t test).
Fig 4: Effect of AMF gene silencing on EC cells growthA. Growth curve by MTS assay. Cells were seeded at low density (2000 per well) and grown for 5 d. Fresh medium was provided everyday (Points, mean of triplicate determinations; bars, SD). B. Cell cycle profile was analyzed by fluorescence-activated cell sorting (FACS); depletion of AMF in endometrial cells altered the cell cycle by arresting the G0/G1 to S phase transition. The summary graphs were presented below the cell cycle profiles. Data represent the mean ± S.D. of three independent experiments (*P < 0.05). C. Ishikawa/shAMF-1, HEC-1B/shAMF-1 and the spheroid formation of the controls in 3D culture was photographed at day 14 in culture (representative images are shown; 400× for the inserts, 200× for all others; scale bar, 100 µm). Quantification of the relative size (lower right) and number (lower left) of spheroids. (*P < 0.05; **P < 0.01)
Fig 5: Scatter plot of the correlation between CSF NLK levels and YKL-40 levels in a subset (n = 47) of the Barcelona aMCI/AD patients and controls. NLK and YKL-40-levels significantly correlated in the combined aMCI/AD and controls (rSP = 0.51, p < 0.0001, gray line) and in the aMCI/AD patients separately (rSP = 0.79, p < 0.0001, black dots and black line), but not in the controls (rSP = 0.033, p = 0.87, white dots, dotted line). Abbreviations: AD, Alzheimer’s dementia; rSP, Spearman rank coefficient; aMCI, amnestic mild cognitive impairment; NLK, neuroleukin; YKL-40, chitinase-3 like-protein 1
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