Fig. 5

Methylglyoxal (MG) stress induces (hyper)activation of the mitogen-activated protein kinase (MAPK) signaling pathway in breast cancer cells. a Transforming growth factor (TGF)βR1, P-SMAD2/3 and SMAD4 protein levels in glyoxalase 1 (GLO1)-depleted MDA-MB-231 cells. Cells were treated with TGFβ 2.5 ng/ml for 2 h where indicated. b P-MEK1/2, MEK2, P-ERK and ERK expression in GLO1-silenced MDA-MB-231 and MDA-MB-468 cells cultured without serum for 24 h. c P-MEK1/2, MEK2, P-ERK and ERK protein levels in MDA-MB-231 and MDA-MB-468 cells treated with MG at the indicated concentrations for 3h in serum-free medium. d P-MEK1/2, MEK2, P-ERK and ERK expression in GLO1-silenced MDA-MB-231 and MDA-MB-468 cells treated with carnosine for 24 h in serum-free medium. All immunoblots were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. Western blot is representative of three independent experiments. e Migration ability toward serum of MDA-MB-231 shGLO1 cells pre-treated with MEK inhibitor, U0126 (10 μM, 3 h), was assessed using Transwell filters. Representative filters are shown for each condition. Scale bar represents 400 μm. f Quantification of migration assays of GLO1-silenced MDA-MB-231 cells treated with U0126. g Tenascin C, COL6A3, lumican and COL4A3 messenger RNA (mRNA) levels in GLO1-depleted MDA-MB-231 cells treated with U0126 for 3 h in serum-free medium were assessed by qRT-PCR. Data were analyzed using two-way analysis of variance followed by Bonferroni post-hoc test and are shown as mean values ± SEM of three independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001