Fig 1.
Compound 4f inhibits breast cancer cell viability.
(A) The structure of compound 4f. (B-D) The effect of compound 4f on the viability of the breast cancer cells MCF-7 and MDA-MB-231 and human normal mammary epithelial cell MCF10A by CCK8 assay at the indicated concentration, n = 3. (E, F) Time-dependent of 4f on the viability of the MCF-7 and MDA-MB-231 cells assessed by CCK8 assay. NS, not significant different from 0 group, *significantly different from 0 group. *p<0.05, **p<0.01 and ***p<0.001, n = 3.
Fig 2.
Compound 4f inhibits cells proliferation in MCF-7 and MDA-MB-231 cell lines.
(A-D) The clonogenic assay and the number of colonies formation analyses. (E-H) The EDU incorporation assay on MCF-7 and MDA-MB-231 cells after treated with compound 4f for 48 h. The red fluorescent staining (EDU-positive) and blue fluorescent staining (Hoechst 33258 staining) cells represent the proliferating and total cells, respectively (E, G). Quantification of EDU-incorporating MCF-7 and MDA-MB-231 cells (F, H). *significantly different from 0 group.*p<0.05, **p<0.01 and ***p<0.001, n = 3.
Fig 3.
Induction of cell cycle arrest at the G2 phase by compound 4f in breast cancer cells.
(A-D) Compound 4f increased the proportion of cells in G2 phase of the cell cycle. (E-H) Western blotting assay for the analysis of CDK1 expression in 4f-treated MCF-7 and MDA-MB-231 cells at indicated concentration for 24 h. *significantly different from 0 group. **p<0.01 and ***p<0.001, n = 3.
Fig 4.
Compound 4f induced apoptosis in MCF-7 and MDA-MB-231 cells.
Annexin V-FITC and Propidium Iodide (PI) assay kit were used to detect cell apoptosis, which was analyzed by flow cytometry after the treatment with 4f for 48 h at different concentrations (0, 1.25, 2.5, 5, 10 μM) in MCF-7 (A, B) and MDA-MB-231 (D, E) cells. Western blotting analysis of the expression of apoptosis related proteins in MCF-7 (C) and MDA-MB-231 (F) cells after the treatment with 4f for 24 h. *significantly different from 0 group. **p<0.01 and ***p<0.001, n = 3.
Fig 5.
Compound 4f induces reduction of the MMP in breast cancer cells.
Cells were treated with 4f at a concentration of 0 μM, 2.5 μM, 5 μM, 10 μM in MCF-7 cell line (A) and 0 μM, 1.25 μM, 2.5 μM, 5 μM in MDA-MB-231 cell line (B) for 24 h, the MMP was measured by JC-1. (C, D) The ratio of decreased MMP. *significantly different from 0 group. *p<0.05, **p<0.01 and ***p<0.001, n = 3.
Fig 6.
4f inhibits MEK/ERK and p38 MAPK pathways in breast cancer cells.
Cells were treated with 4f at a concentration of 0 μM, 2.5 μM, 5 μM, 10 μM in MCF-7 cell line (A) and a concentration of 0 μM, 1.25 μM, 2.5 μM, 5 μM in MDA-MB-231 cell line (B) for 24 h, the expression of phosphorlated and total ERK1/2, p38, JNK and MEK1/2 were detected by western blotting assays. All the data repeated for three times.
Fig 7.
GSDME rather than GSDMD is cleaved during cell pyroptosis of breast cancer cells induced by 4f.
(A, B) Full-length GSDMD (GSDMD-F) and GSDMD-C terminal (GSDMD-C) were detected by western blotting in MCF-7 and MDA-MB-231 cells treated with different doses of 4f. (C, E) Full-length GSDME (GSDME-F) and GSDME-N terminal (GSDME-N) were analyzed by western blotting in MCF-7 and MDA-MB-231 cells treated with different doses of 4f. (D, F) LDH release of MCF-7 and MDA-MB-231 cells treated with 4f at the concentration of 0 or 5 μM. All the data repeated for three times.
Fig 8.
z-DEVD-FMK reduces the cleavage of GSDME.
(A, D) Western blotting analysis of full-length GSDME (GSDME-F) and GSDME-N terminal (GSDME-N) in MCF-7 and MDA-MB-231 cells treated by 4f (5 μM) in the presence or absence of z-DEVD-FMK (50 μM). (B, E) Cell viability analysis by CCK8 kit. (C, F) LDH release was measured using LDH cytotoxicity assay kit. All the data repeated for three times.