Fig 2: 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 3: Myrislignan suppressed the progression of GBM in vivo. (a) Representative HE staining of mice brain sections indicated that myrislignan suppressed the growth of GBM. (b) Kaplan-Meier curves of mice survival. (c) The tumor-bearing brains were weighed. Relative weight of tumors in brain was estimated (DMSO/Myris groups—PBS groups). (d, e) Protein levels of p-p65, Slug, and SLC7A11 in mouse tumor were detected by western blot and quantified. The protein samples were extracted from tumor tissues in DMSO and Myris group of intracranial xenograft models. (f) Expression of p-p65, Slug, E-cadherin, and SLC7A11 was evaluated via IHC staining. Scale bar, 50 µm. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig 4: ISX expression induces TWIST1 and Snail1 expression, and promotes EMT AThe expression levels of ISX, TWIST1, Snail1, fibronectin (FN1), VEGF, and E-cadherin (CDH1) mRNA were examined in a ISX-GFP inducible Tet-ON transformants at 8 h after addition of DOX. Data are presented as mean ± SD in graph (P < 0.001 compared with time point 0 h; Student's t-test) of three independent experiments, each performed in triplicate.BWestern blotting analysis of the protein levels of ISX, HIF1a, TWIST1, Snail1, Slug, ZEB1, Bmi1, E-cadherin (E-Cad.), fibronectin, N-cadherin (N-Cad.), vimentin, and VEGF in A549 and H1299 cells with DOX-inducible ISX expression system 8 h after DOX induction.C, DISX transcriptionally activates luciferase activity driven by TWIST1 (C) and Snail1 (D) promoter regions in A549 cells. Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.EChIP analysis of ISX binding to the TWIST1 promoter region in A549 and H1299 cells (10% input of each group was pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.FISX transactivation activity analyzed by luciferase activity driven by the TWIST1 promoter. Data are presented as mean ± SD in graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.GChIP analysis of ISX binding to the endogenous promoters of TWIST1 (-180/+35) and Snail1(-160/+40) in A549 and cells (10% input of each group was pull down and applied to qPCR). Data are presented as mean ± SD in bar graph (P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate.HEffect of forced expression and knockdown of ISX on cell migration (wound healing) measured in A549 cells. ISXi, ISX-specific shRNAi (described in Materials and Methods). Data are presented as mean ± SD in graph (**P < 0.01, ***P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate. Scale bar, 100 µm.IEffect of forced expression and knockdown of ISX on cell invasion (Transwell) activity measured in A549 cells. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test) of three independent experiments, each performed in triplicate. Scale bar, 100 µm. Source data are available online for this figure.

Fig 5: Mechanistic model for ferroptosis induced by myrislignan in GBM. Through inhibiting the phosphorylation of p65 protein, myrislignan suppressed the NF-?B signaling pathway and regulated EMT-related genes including Slug, Snail1, and E-cadherin, eventually inhibiting EMT. Meanwhile, myrislignan depressed system xc - activity and induced lipid peroxidation via the Slug-SLC7A11 signaling pathway. Furthermore, myrislignan combined with RSL3 enhanced the ferroptosis via targeting SLC7A11 and GPX4, respectively, which also controlled the EMT status of GBM cells.