AMOTL2 mediates the effects of MAGI1 on junctions and on p38 signaling. (A) MTT assay (OD 560 nm) representing 2D cell growth of MCF7shMAGI, MCF7shAMOTL2 and MCF7shMAGI1/shAMOTL2 cells as compared to MCF7shLuc. Bars represent mean ± SD (n = 10 wells as replicates) of a representative experiment (n = 3). Unpaired two-tailed Student's t-test; **p < 0.01; ***p < 0.001. (B) Quantification of colony numbers of MCF7 cells (shMAGI1, shAMOTL2 and shMAGI1/shAMOTL2) grown in anchorage independent conditions (soft agar assay) and represented as fold increase compared to MCF7shLuc cells. Data are presented as the means ± SD (n = 3). Unpaired two-tailed Student's t-test; *p < 0.05. Western blot analyses confirmed the 90% knockdown for MAGI1 in MCF7shMAGI1 and MCF7shMAGI1/shAMOTL2 and QPCR data confirmed the 50 to 60% knockdown for AMOTL2 in MCF7shAMOTL2 and MCF7shMAGI1/shAMOTL2 respectively (determined by RT-qPCR and/or Western Blot ; Data not shown). (C) Western blot analysis showing protein expression and/or phosphorylation (activation) of p38 and JNK stress signaling pathways as well as junctions’ components in MCF7shMAGI1 as compared to MCF7shLuc cells when AMOTL2siRNA was transfected. Tubulin was used as a loading control. Western blot experiments have been repeated at least three times. Uncropped blots can be found in the Supplementary Information. (D) Quantification of the representative Western blot showing protein expression and represented in panel C. Phosphorylated proteins were quantified as compared to their total protein counterparts to evaluate their activation and junctions’ proteins were quantified as compared to tubulin that was used as a loading control. Upper: Phosphorylated p38/Total p38 protein quantification and Lower: protein/Tubulin quantification. (E) Model for the role of MAGI1 during Luminal BCa. MAGI1 prevents the accumulation of junctional AMOTL2 and E-cadherin as well as ROCK activity thus releasing cellular stiffness. Increased AMOTL2 and ROCK then activate p38 stress signaling responsible for the increased tumorigenicity of MAGI1-deficient cells. Anti and Pro tumorigenic events are highlighted in green and red respectively.