Figure 5. MG induces YAP co-transcriptional activity in breast cancer cells.

(A) Stable knockdown of GLO1 (shGLO1#2) in MDA-MB-231 results in upregulation of several YAP target genes (ANKFN1, RIMS3, KCNK1, EMP2, OSBP2, IRAK3, WTN5A and CTGF) at the mRNA level as assessed by qRT-PCR. Silencing of YAP using two independent siRNAs (siYAP#1 and #2, 48 hr post-transfection) significantly reversed YAP target genes induction in GLO1 depleted cells. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SD of one representative experiment (n = 4). (B) Chromatin immunoprecipitation of YAP at the CTGF promoter in sparse and confluent MDA-MB-231 cells treated or not with MG. TEAD PCR primers, and not control primers, target TEAD binding site on CTGF promoter (see sequences under 'Materials and methods' section). The use of TEAD primers indicated that YAP was present at the CTGF promoter in sparse cells (positive control) and in confluent MG-treated cells when compared to untreated confluent cells. Data were analyzed using one-way ANOVA followed by Newman-Keuls post-test and shown as the mean values ± SEM of three independent experiments. (C) CTGF mRNA level assessed by qRT-PCR in MDA-MB-231 cells treated with MG 300 µM until confluence and then with TGFβ 2.5 ng/ml during 2 hr. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of five independent experiments. (D) MG-mediated CTGF induction in presence of TGFβ is not observed upon YAP silencing (siYAP#1 and #2) when compared to control (siGl3) cells. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. (E) YAP (Santa Cruz antibody, H125) and TEAD1 IF co-localization in MDA-MB-231 cells cultured under low (Sparse) density used as positive control and in high-density cultured cells (Confluent) in presence of MG. Magnification 630x. Data are representative of three independent experiments. (F) DNA quantification assay showing an increased proliferation of GLO1-silenced MDA-MB-231 (shGLO1#1 and #2) compared to control (shNT) at 72 hr. Silencing of YAP (siYAP#1 and #2) reversed this effect. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of four independent experiments. (G) Validation of YAP silencing by Western blot in MDA-MB-231 shGLO1 cells after 72 hr related to panel F and Figure 5—figure supplement 2F. *p<0.05, **p<0.01, ***p<0.001 and ns = not significant.

DOI: http://dx.doi.org/10.7554/eLife.19375.014

Figure 5—figure supplement 1. Inverse correlation between GLO1 and YAP target genes expression.

(A) Validation of YAP silencing (siYAP#1 and #2) in shGLO1 MDA-MB-231 cells 48 hr post-transfection by qRT-PCR. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SD of one representative experiment (n = 4). ** p<0.01 and *** p<0.001 (B) Inverse correlation between the expression of GLO1 and 12 representative YAP target genes in breast cancer patients (n = 103). R_p: Pearson correlation coefficient.

DOI: http://dx.doi.org/10.7554/eLife.19375.015

Figure 5—figure supplement 2. MG induces YAP co-transcriptional activity in breast cancer cells.

(A) Western blot detection of YAP in MDA-MB-231 cells under the indication conditions. Immunoblot is representative of three independent experiments. (B) Western blot of Phospho-Smad2/3 and Smad2/3 in MDA-MB-231 treated with MG until confluence and then with TGF-β during 2 hr. β-actin is used for normalization. (C and D) YAP mRNA and protein level assessed by qRT-PCR and Western blot, respectively, in MDA-MB-231 cells silenced for YAP (siYAP#1 and #2) and treated in the same conditions as in Figure 5D. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. (E) YAP (Cell Signaling, 4912) and TEAD1 IF co-localization in MDA-MB-231 cells cultured under low (Sparse) density used as positive control and in high-density cultured cells (Confluent) in presence of MG. Magnification 630x. Data are representative of two independent experiments. (F) Proliferation assay on GLO1-depleted MDA-MB-231 (shGLO1#1 and #2) silenced or not for YAP (siYAP#1 and #2) at different time points. Data (72 hr) were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of three independent experiments. All immunoblots are representative of three independent experiments. ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.19375.016

Figure 5—figure supplement 3. MG increases YAP-mediated migratory potential in breast cancer cells.

(A) E-cadherin and vimentin EMT markers are down-regulated and up-regulated, respectively, upon MG treatment in MDA-MB-468 breast cancer cells as shown by western blot. β-actin is used for normalization. (B) High-density MDA-MB-468 cells treated with MG demonstrated reduced and disrupted E-cadherin junctions using immunofluorescence. Magnification 630x. Zoomed pictures are shown where indicated. Data are representative of three independent experiments. (C) MDA-MB-468 cells treated with MG until confluence showed a higher migratory capacity compared to control cells as assessed by wound healing assay (16 hr) and not in YAP-silenced conditions. Data were analyzed using two-way ANOVA followed by Bonferroni post-test and shown as the mean values ± SEM of five independent experiments. (D) Validation of YAP silencing in MDA-MB-468 in the same conditions than panel C. ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.19375.017