Figure 5 | Scientific Reports

Figure 5

From: MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis

Figure 5

The loss of MAGI1 tumorigenic phenotypes are not associated with active YAP. (A) Western blot analysis on whole protein extracts (n = 3) of phosphorylated YAP (S397 and S127), total YAP and TAZ, phosphorylated LATS1, total LATS1, phosphorylated MOB1, non-phosphorylated (active) β-catenin, total β-catenin and MAGI1 in MCF7shMAGI1 cell lines compared to MCF7shLuc. Tubulin was used as a loading control. Uncropped blots can be found in the Supplementary Information. (B) Representative immunofluorescence images (n = 3) of endogenous MAGI1 and YAP protein expression in MCF7shLuc and MCF7shMAGI1 cells cultured to similar confluence (60.4% and 63.2% respectively). Scale bar = 10 µm. (C) Quantification of MAGI1 and YAP staining in a tissue micro-array (TMA) from luminal breast cancer patients in a cohort of 28 patients, showing the strong correlation between MAGI1 membranous and YAP nuclear localizations; Fisher exact test, ***p < 0.001. Histogram shows the percentages of patients having a nuclear or a cytoplasmic YAP staining according to the absence or the presence of MAGI1 membranous staining. (D) Real-time qRT-PCR showing the expression of canonical Hippo pathway target genes (CTGF, CYR61, BIRC2, AXL and AREG) as well as AMOTL2 in MCF7shMAGI1 compared to the control MCF7shLuc cells and normalized with GAPDH expression. Data are presented as the means ± SD (n = 3). Unpaired two-tailed Student's t-test; *p < 0.05; **p < 0.01; ***p < 0.001. (E) Real-time qRT-PCR showing the expression of two distinct Wnt pathway target genes (AXIN2 and CCND1) in MCF7shMAGI1 compared to the control MCF7shLuc cells and normalized with GAPDH expression. Data are presented as the means ± SD (n = 3). Unpaired two-tailed Student's t-test revealed no statistical differences. (F) WNT reporter activity assay showing the TOP/FOP ratio in MCF7shMAGI1 as compared to MCF7shLuc control cells. Data are presented as the means ± SD (n = 3). Unpaired two-tailed Student's t-test revealed no statistical differences.

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