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Fig. 2 | Journal of Experimental & Clinical Cancer Research

Fig. 2

From: Methylglyoxal: a novel upstream regulator of DNA methylation

Fig. 2

MG stress induces the overexpression of DNMT3B that is reversed using MG scavengers and that mediates enhanced migratory capacity of GLO1-depleted cells.A Among DNMTs, endogenous MG stress consistently increased DNMT3B protein levels across GLO1-depleted (shGLO1 #1 and #2) MDA-MB-231 and Hs578T TNBC breast cancer cells as assessed using western blot on total protein cell extracts and compared with control (shNT) cells. Additionally, exogenous MG treatment significantly up regulated DNMT3B levels in both breast cancer cell lines $, †. B Among DNMTs, expression of DNMT3B was elevated in shGLO1 (n = 6) when compared with shNT mouse xenografts (n = 3), as assessed using western blot on total protein tumor extracts †. C DNMT3B protein abundance, in presence of cycloheximide (10 μg/mL) at the indicated timing, in shNT MDA-MB-231 cells demonstrated shorter DNMT3B half-life contrasting with GLO1-depleted cells. Data are represented as means ± SEM of three independent experiments and were analyzed using two-way analysis of variance (ANOVA) followed by Dunnett test (**** p < 0.0001). Corresponding western blots are shown in Fig. S2C. D and E Carnosine (48 h) and aminoguanidine (24 h) treatments significantly reduced DNMT3B protein expression in a dose-dependent manner in GLO1-depleted MDA-MB-231 cells, respectively $, †. F Carnosine re-induces the expression of metastasis-related TSGs under study in MDA-MB-231 cells as assessed using RT-QPCR §. G The migratory capacity of GLO1-depleted MDA-MB-231 cells was evaluated upon 5-AZA treatment (72 h) using a scratch wound assay under Incucyte® life cell microscopy. Results are given as a percentage of relative wound closure over time for shGLO1#2 $, ¥. H Relative wound closure at 8 h time point post scratch in shGLO1#2 cells treated with increasing doses of 5-AZA $, ‡. I Representative pictures illustrating the wound closure at 8 h post scratch of MDA-MB-231 shGLO1#2 cells silenced (siDNMT3B) or not (Irr siRNA) for DNMT3B and compared to shNT cells. J Migratory capacity (8 h time point) of MDA-MB-231 shGLO1#2 cells upon DNMT3B silencing $, ‡. $ One of three experiments is shown. † Alpha-tubulin was used as a loading control. ‡ Data represent the mean values ± SD of three technical replicates and were analyzed using a one-way analysis of variance (ANOVA) followed by a Dunnett test (** p < 0.01, *** p < 0.001, **** p < 0.0001). § Data represent the mean values ± SEM of three independent experiments and were analyzed using a one-way analysis of variance (ANOVA) followed by a Dunnett test (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns: not significant). ¥ Data represent the mean values ± SD of three technical replicates and were analyzed using a two-way analysis of variance (ANOVA) followed by a Dunnett test (**** p < 0.0001)

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