Fig. 6

(Hyper)activation of mitogen-activated protein kinase kinase (MEK) signalization leads to SMAD1 phosphorylation and regulation of methylglyoxal (MG) stress pro-metastatic signature genes in breast cancer cells. a SMAD1 immunofluorescence staining in glyoxalase 1 (GLO1)-silenced MDA-MB-231 cells cultured in serum-free conditions. Data are representative of three independent experiments. Magnification × 630. Zoomed pictures (white square) are shown where indicated. b P-SMAD1 (ser206), P-SMAD1/5 (ser463/465), SMAD1 and SMAD5 protein levels in GLO1-silenced MDA-MB-231 and MDA-MB-468 cells cultured without serum for 24 h. c SMAD1 and SMAD5 mRNA levels in GLO1-depleted MDA-MB-231 cells were assessed by qRT-PCR. Data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett post-hoc test and shown as mean values ± SEM of three independent experiments. d P-SMAD1 (ser206), P-SMAD1/5 (ser463/465), SMAD1 and SMAD5 protein level in GLO1-silenced MDA-MB-231 and MDA-MB-468 cells treated with MG at the indicated concentrations for 3 h in serum-free medium. e (P-)ERK and (P-)SMAD1 (ser206) protein level in GLO1-silenced MDA-MB-231 cells treated with U0126 MEK inhibitor (10 μM, 3 h) in serum-free medium. f SMAD1 target genes mRNA levels were assessed using qRT-PCR in SMAD1-silenced MDA-MB-231 shGLO1 cells. SMAD1 mRNA level was assessed to validate efficient SMAD1 siRNA silencing. g Migration ability toward serum of SMAD1-silenced MDA-MB-231 shGLO1 cells was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. h Quantification of migration assays of SMAD1-silenced MDA-MB-231 shGLO1 cells. i SMAD1 protein level assessed by immunoblot to validate SMAD1-silencing using siRNAs in GLO1-depleted MDA-MB-231 cells (related to panels g and h). Immunoblot data were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. All western blots are representative of three independent experiments. Data were analyzed using two-way ANOVA followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001