Fig. 3

The proliferation and migration of MDA-MB-231 cells were inhibited by M1-21. A-B MDA-MB-231 cells were treated with M1-21 or M1-21mut (20 µM) for 24 h and total RNA samples were prepared for RNA sequencing. Gene set enrichment analysis (GSEA) was performed to show inhibition of the genes of DNA replication initiation genes (A) and cell adhesion gene activation genes (B). C qPCR was used to verify changes in PCNA and CyclinB1 mRNA levels from panel A samples. GAPDH was used as loading control. D MDA-MB-231 cells were treated as panel A and cell lysates were prepared. PCNA and CyclinB1 protein levels were measured by Western blotting. β-actin was used as a loading control. E MDA-MB-231 cells (200 cells/well) were treated with M1-21 or M1-21mut (20 µM) for 14 days. The cells were fixed with 4% paraformaldehyde and stained with crystal violet for imaging. The treatment’s ability to form colonies was measured by counting the number of cell colonies in each well. F BALB/c nude mice (female, 6 weeks old) were subcutaneously injected with MDA-MB-231 cells (1 × 10^6 cells/mouse). One week later, the mice were randomly divided into two groups followed by intraperitoneal PBS injection (Control, n = 6) or M1-21 (30 mg/kg, n = 6) once every two days for 19 days. The volume of engrafted tumors was measured every two days and growth curves were obtained at the end of the experiment. The tumor volume (V) was calculated by: V = length × diameter² × 1/2. G Images and weight of engrafted tumors on Day 19. H MDA-MB-231 cells were seeded into plates to reach 90% confluence. A 200 µl pipette tip was used to thread a line and then photographed. Cells were then treated with M1-21 (10 µM) or M1-21mut (10 µM) and cell migration was recorded at 36 h post-wound formation. Number of repetitions = 3. I-J MDA-MB-231 cells were treated with M1-21 (10 µM) or M1-21mut (10 µM) and 24 h later cells were harvested for the preparation of total RNA or cell lysates. The levels of mRNA or protein of E-cadherin (E-cad), Vimentin (VIM), and N-cadherin (N-cad) were measured by qPCR or Western blotting, respectively. GAPDH or β-actin was used as a loading control for mRNA or protein. mRNA values represented the mean ± SD of three replicates, and significance was calculated using unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001