Fig 2: ZNF471 attenuates the epithelial-mesenchymal transition (EMT) and suppresses cell stemness in breast cancer. a Morphological changes of MB231 cells after transfected with vector and ZNF471. b Expression of E-cadherin, N-cadherin, Vimentin, Snail, Slug, and MMP1,3 by western blot assay in empty vector cells and ZNF471 overexpressing cells. c Western blot was performed to examined N-cad, vimentin, slug, snail and MMP1 in BT549 cells. d qPCR analysis of stem cell markers expression (Data are presented as the mean ± SD; *p < 0.05; **p < 0.01; ***p < 0.001)

Fig 3: MMP1 expression and promoter activity increases in HCT116 colon cancer cells treated with the antiplatelet agents. (A) HCT116 cells were treated for 24 h with DMSO (as a control), Asp (1000 µM), Cpg (100 µM), Psg (100 µM), or Tcg (20 µM). MMP1 mRNA and protein expressions were assessed by qRT-PCR and Western blotting. MMP1 protein band intensity was quantified by density using ImageJ software. The bar graph shows the relative expression density of MMP1 normalized to GAPDH levels. (B) LoVo and SNU398 cells were treated for 24 h with DMSO, Asp, Cpg, Psg, or Tcg. MMP1 mRNA and protein expressions were assessed by qRT-PCR. (C) HCT116 cells were transfected with SERPINE1 siRNA (siSERPINE1) and SERPINE1 overexpression vector (pSERPINE1) for 24 h. qRT-PCR was performed to detect the mRNA levels of SERPINE1 and MMP1. (D) Upper panel shows a schematic representation of the MMP1 promoter region with the luciferase gene in the pGL3 vector. The antiplatelet agents were applied to HCT116 cells containing the SERPINE1 promoter vector and measured using a luciferase assay. In (A-D), data are shown as means ± SD. Statistical significance was determined using ordinary two-way ANOVA and Student’s t-tests; ns, not significant; *, p < 0.05; **, p < 0.01 and ***, p < 0.001; n = 3 per group.

Fig 4: PHI treatment activates PPAR? signaling by downregulating MMP8 expression. (A and B) Following MMP8 overexpression, the induction of cell apoptosis of LPS-treated BEAS-2B cells, which were pretreated with PHI, was assessed using a TUNEL assay (magnification, ×200; scale bar, 100 µm). ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ???P<0.001 vs. LPS + PHI group. (C) Western blotting was used to determine the expression levels of the apoptosis-associated proteins in LPS-induced BEAS-2B cells treated with PHI and transfected with pcDNA3.1-MMP8. ***P<0.001 vs. control; ##P<0.01, ###P<0.001 vs. LPS group; ??P<0.01 vs. LPS + PHI group. (D) Western blotting was used to determine the expression levels of PPAR? in LPS-induced BEAS-2B cells treated with PHI and transfected with pcDNA3.1-MMP8. ***P<0.001 vs. control; ###P<0.001 vs. LPS group. PHI, phillygenin; PPAR?, peroxisome proliferator-activated receptor ?; LPS, lipopolysaccharide; NC, negative control; c-, cleaved.

Fig 5: PHI treatment suppresses inflammation of LPS-induced BEAS-2B cells. pcDNA3.1-MMP8 was constructed and the expression levels of MMP8 were measured via (A) reverse transcription-quantitative PCR and (B) western blot analyses. ***P<0.001 vs. control and pcDNA3.1-NC. (C-E) Following transfection of pcDNA3.1-MMP8 into LPS-induced BEAS-2B cells pretreated with PHI, the expression levels of the inflammatory factors were measured via ELISA. ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ???P<0.001 vs. LPS + PHI group. (F) Following transfection of pcDNA3.1-MMP8 into LPS-induced BEAS-2B cells pretreated with PHI, the expression levels of the inflammation-associated factors were measured via western blotting. ***P<0.001 vs. control; ###P<0.001 vs. LPS group; ?P<0.05, ???P<0.001 vs. LPS + PHI group. PHI, phillygenin; LPS, lipopolysaccharide; NC, negative control; cox-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.