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. 2016 Oct 19;5:e19375. doi: 10.7554/eLife.19375

Figure 2. Methylglyoxal induces YAP persistence in confluent breast cancer cells.

(A) Immunofluorescence (IF) staining shows that YAP (Santa Cruz antibody, H125) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density (Confluent) in MDA-MB-231 cells. In contrast, cells treated with increasing doses of MG until they reach confluence showed significant YAP cellular accumulation. Zoomed pictures are shown where indicated. Magnification 630x. Data are representative of three independent experiments. (B) Quantification of panel A experiment reports the intensity of YAP staining that colocalized with DAPI staining as described in 'Materials and methods' section. Nuclear YAP IF staining intensity shows a significant dose-dependent increase in presence of MG. Data were analyzed using one-way ANOVA followed by Dunnett post-test and shown as the mean values ± SEM of three independent experiments. (C) YAP, P-YAP (S127 and S381) and TAZ expression in MDA-MB-231 cells treated with MG (300 µM) until they reached confluence using western blot. Immunoblot data were quantified by densitometric analysis and normalized for β-actin. Numbers represent fold increase relative to the condition shown with bold number. (D) MDA-MB-231 cells cultured until they reached high density and treated concomitantly with MG (300 µM) and carnosine (10 mM), a MG scavenger, impeded cellular accumulation of YAP. Magnification 630x. Data are representative of three independent experiments. (E) Quantification of panel D experiment. Data were analyzed using one-way ANOVA followed by Bonferroni post-test and are shown as the mean values ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 and ns = not significant.

DOI: http://dx.doi.org/10.7554/eLife.19375.003

Figure 2.

Figure 2—figure supplement 1. Methylglyoxal induces YAP accumulation in confluent breast cancer cells.

Figure 2—figure supplement 1.

(A and D) Immunofluorescence staining shows that YAP (Santa Cruz antibody, H125) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density (Confluent) in MDA-MB-468 and MCF7 cells. In contrast, cells treated with increasing doses of MG until they reach confluence showed YAP cellular accumulation. Magnification 630x. Zoomed pictures are shown where indicated. Data are representative of three independent experiments. (B and E) Quantification of panel A and C experiments reports the intensity of YAP staining that colocalized with DAPI staining as described in 'Materials and methods' section. Nuclear YAP IF staining intensity shows a significant dose-dependent increase in presence of MG. Data were analyzed using one-way ANOVA followed by Dunnett post-test and shown as the mean values ± SEM of three independent experiments. (C and F) YAP, P-YAP (S127 and S381) and TAZ expression in MDA-MB-468 and MCF7 cells treated with MG (300 µM) until they reach confluence using western blot. Immunoblot data were quantified by densotimetric analysis and normalized for β-actin. Data are representative of three independent experiments. (G) qRT-PCR analysis of YAP gene in the indicated breast cancer cells treated with MG. Data were analyzed using unpaired student’s t test for each cell line and shown as the mean values ± SEM of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.
DOI: http://dx.doi.org/10.7554/eLife.19375.004
Figure 2—figure supplement 2. Methylglyoxal induces YAP accumulation in confluent breast cancer cells.

Figure 2—figure supplement 2.

(A, B and C) Immunofluorescence (IF) staining shows that YAP (Cell Signaling antibody, 4912) is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density (Confluent) in MDA-MB-231, MDA-MB-468 and MCF7 cells. In contrast, cells treated with increasing doses of MG until they reach confluence showed significant YAP cellular accumulation. Magnification 630x. Data are representative of three independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.19375.005
Figure 2—figure supplement 3. Methylglyoxal induces TAZ accumulation in confluent breast cancer cells.

Figure 2—figure supplement 3.

(A, B, and C) Immunofluorescence (IF) staining shows that TAZ is mainly localized in the nucleus at low cellular density (Sparse) and is weakly detectable at high cellular density (Confluent) in MDA-MB-231, MDA-MB-468 and MCF7 cells. In contrast, cells treated with increasing doses of MG until they reached confluence showed significant TAZ cellular accumulation. Magnification 630x. Data are representative of two independent experiments.
DOI: http://dx.doi.org/10.7554/eLife.19375.006