Fig 2: PTX3 expression in breast cancer cells is associated with stem-like features and epithelial-mesenchymal transition. (A) Tumorsphere formation of MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with siPTX3 or rhPTX3 for 14 days, and tumor spheres expansion were analyzed at 40x magnification under a microscope (bar = 50 µm; magnification, 400x). (B) PTX3 promotes cell migration and invasion in breast cancer. MCF-7 and MDA-MB-231 cells were treated with either siPTX3 or rhPTX3. The migration and invasion abilities of the cells were examined by migration and invasion assay (Transwell assay). (C,D) Effect of PTX3 on stem-like features and epithelial-mesenchymal transition markers. siPTX3 or rhPTX3-treated MDA-MB-231 and MCF-7 cells were lysed and subjected to 12% SDS-PAGE and analyzed by western blotting with antibodies against PTX3, Oct4, Sox2, E-cadherin, N-cadherin, and Snail. GAPDH was used as an internal control. The data was statistically significant at *P < 0.05; **P < 0.01; and ***P < 0.001 as compared to control. Data are represented as mean ± SEM of three independent experiments.

Fig 3: BmK AGAP inhibits epithelial-mesenchymal transition (EMT) of breast cancer cells. (A) Relative gene expression of N-cadherin and E-cadherin in MCF-7 and MDA-MB-231 cells treated with different concentrations of rBmK AGAP. GAPDH served as internal control. (B) Protein expression levels of Snail1, N-cadherin, and E-cadherin following rBmK AGAP treatment of MCF-7 and MDA-MB-231 cells. The cells were treated for 48 h, lysed, subjected to SDS-PAGE and analyzed by western blotting with antibodies against Snail1, N-cadherin, and E-cadherin. (C,D) Immunofluorescence assessment of cadherin and N-cadherin in MCF-7 and MDA-MB-231 cells following rBmK AGAP treatment. MCF-7 and MDA-MB-231 cells were stained with anti- E-cadherin and N-cadherin antibodies (red) following rBmK AGAP treatment and were observed under the fluorescence microscopy. DAPI was used for nuclear staining (bar = 50 µm; magnification, 400x). (E,F) rBmK AGAP inhibits the migration and invasion potentials of MCF-7 and MDA-MB-231 cells. MCF-7 and MDA-MB-231 cells were treated with different concentrations of rBmK AGAP and examined by migration and invasion assay. The data was statistically significant at *P < 0.05; **P < 0.01; and ***P < 0.001 as compared to untreated cells. Data are represented as mean ± SEM of three independent experiments.

Fig 4: Myrislignan regulated the expression of EMT-related genes and inhibited the phosphorylation of p65 protein. (a–d) The protein (E-cadherin, Snail1, and Slug) expression was detected via western blot assay after myrislignan treatment in U87 and U251 cells. (e–j) The protein (total p65/I?B-a and phosphorylated p65/I?B-a) levels were measured via western blot assay after myrislignan treatment in U87 and U251 cells. (k) Immunofluorescence of p65 and p-p65 protein after myrislignan treatment in U87 cells. (l, m) Dual-luciferase reporter assay was conducted to evaluate the status of NF-?B signal in GBM cells treated with myrislignan. Scale bar, 10 µm. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no significance.

Fig 5: Myrislignan regulated the ferroptosis of GBM cells via Slug-SLC7A11 pathway. (a) Protein (SLC7A11, Snail1, and p-p65) levels were detected via western blot assay after myrislignan treatment with transfection of HA-Snail1 in U87 cells. (b, c) Protein (SLC7A11, Slug, and p-p65) levels were detected via western blot assay after myrislignan treatment with transfection of Flag-Slug or sh-Slug in U87 cells. (d–g) The lipid ROS assay, MDA assay, glutathione assay, and Cys assay were conducted to measure the levels of lipid ROS, MDA, GSH, and Cys in U87 cells after myrislignan treatment with transfection of Flag-Slug, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ns: no significance.