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Fig. 4 | Breast Cancer Research

Fig. 4

From: Methylglyoxal, a glycolysis metabolite, triggers metastasis through MEK/ERK/SMAD1 pathway activation in breast cancer

Fig. 4

Dicarbonyl stress promotes migration and invasion of breast cancer cells. a Migration ability of glyoxalase 1 (GLO1)-depleted MDA-MB-231 toward serum was assessed using Transwell filters. Where indicated, cells were pre-treated with the methylglyoxal (MG) scavengers, carnosine and aminoguanidine, 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. b Quantification of migratory ability of GLO1-silenced MDA-MB-231 cells treated with carnosine and aminoguanidine. Data were analyzed using two-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test and shown as mean values ± SEM of three independent experiments. c E-cadherin and vimentin expression in MCF7 and MCF7-M cells. β-actin protein level was used as loading control. A representative western blot of two independent experiments is shown. d Extracellular acidification (ECAR) and oxygen consumption (OCR) rates in MCF7 and MCF7-M cells using Seahorse flux analyzer. G, O and 2-DG correspond to injection of glucose, oligomycin and 2-deoxyglucose, respectively. Data were analyzed using two-way ANOVA and are representative of two independent experiments. e Glucose uptake was assessed in MCF7 and MCF7-M cells using FACS analysis. Data were analyzed using Student’s t test and are shown as mean values ± SEM from three independent experiments. f MG-Hs and argpyrimidine MG adducts levels were detected by immunoblot using specific antibodies in MCF7 and MCF7-M cells, with β-actin as loading control. g GLO1 and Nrf2 expression in MCF7 and MCF7-M cells. β-actin protein is used as loading control. Western blot is representative of three independent experiments. h GLO1 maximal activity was measured in MCF7 and MCF7-M cells and expressed as arbitrary units (A.U.) per mg of proteins. Data were analyzed using Student’s t test and are shown as mean values ± SEM of three independent experiments. i Migration ability toward serum of MCF7 and MCF7-M cells was assessed using Transwell filter. Cells were pre-treated with carnosine and aminoguanidine MG scavengers for 24 h prior to the assay. Representative filters are shown for each condition. Scale bar represents 400 μm. j Quantification of MCF7 and MCF7-M cells migration assays. Data were analyzed using two-way ANOVA 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

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