Fig 1: SFRP2 leads to decreased activity of Wnt signaling in AtT20 cells. (A) Overexpression of SFRP2 reduced the levels of β-catenin both in the cytoplasm and nucleus. (B) SFRP2 overexpression reduced the expression of c-myc and β-catenin, and also decreased the phosphorylation but not effect on total of Erk in AtT20 cells. (C) SFRP2 treatment decreased the relative mRNA level of c-myc. *P<0.05 and **P<0.01 vs. Con or 0 nM. Con, control; SFRP2, secreted frizzled-related protein 2; p-, phospho-; Erk, extracellular signal-regulated kinase.
Fig 2: SFRP2 inhibits AtT20 cell migration. (A) Representative images of the wound healing assay in the SFRP2-transfected or vector-transfected AtT20 cells at 30 h. (B) Relative expression of MMP-2, MMP-7 and MMP-9 in two groups. *P<0.05 and **P<0.01. Con, control; SFRP2, secreted frizzled-related protein 2; MMP, matrix metallopeptidase.
Fig 3: SFRP2 decreases the proliferation and clonogenic ability of AtT20 cells in vitro. (A) Reverse transcription-quantitative polymerase chain reaction and (B) western blot analysis confirmed increased expression of SFRP2 in stably transfected AtT20 cells. (C) Cell proliferation was measured with the CCK-8 assay from 24 to 66 h. (D) Colony formation and the quantitative representation. (E) Expression of PCNA and cyclin E determined by western blot analysis in SFRP2-transfected AtT20 cells compared with controls. (F) Following SFRP2 treatment, AtT20 cell viability was detected from 24 to 72 h. (G) Flow cytometric analysis and the ratio of apoptosis (upper right quadrant, advanced apoptosis rate; the lower right quadrant, early apoptosis) between SFRP2-transfected and vector-transfected AtT20 cells. *P<0.05, **P<0.01 and ***P<0.005 vs. Con. Con, control; SFRP2, secreted frizzled-related protein 2; PCNA, proliferating cell nuclear antigen.
Fig 4: Correlation analysis between SFRP2 expression and clinical phenotype in CD. Relative expression of SFRP2 in (A) microadenoma (n=19) or macroadenoma (n=4) of CD, and (B) in non-invasive (n=21) or invasive (n=5) corticotroph adenoma. Correlation of SFRP2 expression and hormone parameters in CD; (C) plasma ACTH, (D) urinary free cortisol, (E) midnight serum cortisol. *P<0.05 and **P<0.01. CD, Cushing's disease; SFRP2, secreted frizzled-related protein 2; ACTH, adrenocorticotrophic hormone.
Fig 5: Overexpression of SFRP2 attenuates ACTH secretion in vitro. (A) Measurement of ACTH secretion in SFRP2-transfected cells and controls. (B) Variation in ACTH secretion following treatment with SFRP2 at different concentrations. Reverse transcription-quantitative polymerase chain reaction confirmed decreased levels of (C) POMC and (D) PTTG1 mRNA in stable SFRP2 transfected cells. *P<0.05, **P<0.01 and ***P<0.005 vs. Con or 0 nM. ATCH, adrenocorticotrophic hormone; Con, control; SFRP2, secreted frizzled-related protein 2; POMC, pro-opiomelanocortin-α; Pttg1, pituitary tumor-transforming gene 1.
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