Fig 2: Myocyte enhancer factor 2C (MEF2C) expression in human brain metastases from triple-negative breast carcinoma. Double labeling immunofluorescence analysis was performed for MEF2C with pan-cytokeratin in sections from resected brain metastasis, from triple-negative breast cancer patients. MEF2C shows colocalization with pan-cytokeratin. The different channels of the labeling are presented, as well as the merged pictures. Hoechst is labeling the nucleus. Representative photographs from four different cases analyzed are shown.

Fig 3: MiR-96-5p suppresses the expressions of myogenic factors and FHL1. C2C12 myoblasts were transfected with 100 nM of scRNA control, siFHL1, miR-96-5p mimic, or antimiR-96-5p. (A) Representative immunoblots obtained after differentiation for three days. (B) Quantitative analysis of FHL1 expression. (C–F) Quantitative analysis of MyoD, MyoD, MEF2C, and MyHC expressions. Expression levels were normalized vs. ß-Actin. The values are expressed as the relative ratio, where the intensity of normalized scRNA control was set to one. Results are expressed as means ± SEMs (n > 3). ***, p < 0.001 vs. scRNA.

Fig 4: Myocyte enhancer factor 2C (MEF2C) expression in the brain parenchyma along brain metastasization. Immunofluorescence analysis of MEF2C in brain sections for different timepoints after 4T1 or vehicle (control) injection. Double labeling with claudin-5 showed that MEF2C is not expressed in endothelial cells but only in perivascular cells (A). Double labeling immunofluorescence analysis of MEF2C and pan-cytokeratin revealed that MEF2C-positive cells are tumor cells (B). MEF2C labeling was also observed in other star-shaped cells (arrows) (B). Semiquantitative analysis of MEF2C expression along time showed an increasing expression in tumor cells (C) and an increasing nuclear translocation of MEF2C (D). Results are expressed as mean ± SEM. A one-way ANOVA, followed by the Bonferroni post hoc test, was used to evaluate the significant changes in the parameters between the different timepoints. ## P < 0.01 between indicated groups.

Fig 5: Summary of the effects of molecule (mol) 25 and mol 27 in highly metastatic triple-negative breast cancer (TNBC) cells. The exposure of TNBC cells to mol 25 promoted the expression and activation of cyclin D2, in line with cell cycle inhibition. Moreover, mol 25 inhibited Ki-67 and forkhead box P1 (FOXP1) expression at the cell nuclei, decreasing the TNBC cell’s proliferation and migration abilities. An inhibitory effect in cytoplasmic myocyte enhancer factor (MEF)2C expression was also observed, pointing to a decrease in cellular processes such as cell invasion. On the other hand, mol 27 presented inhibitory effects towards Ki-67 and MEF2C, mainly affecting invasive/migratory and proliferative properties of malignant cells, with the cell cycle appearing to be inhibited, as observed by the increased expression of cyclin D2. Together, both mol 25 and 27 appear as strong candidates to tackle TNBC. Arrows indicate the inducer (up) or inhibitor (down) potential of each mol in the cellular events; the number of arrows increases with the stronger effect of mol 25 and 27. (-) indicates that no effect was observed or conclusive.