Figure 3

Retinoic acid-dependent differentiation of SKNBE2 cells decreases metformin antiproliferative effects. A) Morphological analysis of SKNBE2 cell lines treated with retinoic acid (RA) 10 μM or vehicle (scale bar = 100 μm). B) Cell counting assay in SKNBE2 differentiated with 10 μM retinoic acid (RA) and the respective control, in the presence or absence of metformin (Met). The number of cells, are reported as Met/CTR ratio. C) Cell death analysis by FACS analysis for SYTOX blue staining. The percentage of cell death is reported as Met/CTR ratio. D) Morphological analysis of SKNBE2 cells treated with 1 μM retinoic acid (RA) or vehicle (scale bar = 200 μm). E) Cell counting assay of SKNBE2 cells treated with 1 μM retinoic acid (RA) and the respective control, in the presence or absence of metformin (Met). The number of cells, are reported as Met/CTR ratio. F) Western blot analysis of AMPk and mTOR phosphorylation/activation induced by metformin (Met) before and after the induction of RA-mediated differentiation of SKNBE2 cells. Equal loading of proteins was ensured by α-tubulin expression. G) Morphological analysis of SHSY5Y cells treated with retinoic acid (RA) 1 μM or vehicle (scale bar = 100 μm). H) Cell counting assay in SHSY5Y cells, treated with retinoic acid (RA) 1 μM and the respective control, in the presence or absence of metformin (Met). The number of cells, are reported as Met/CTR ratio. Images were taken by EVOS fl, AMG. (*p < 0.05; **p < 0.01; ***p < 0.001).