Fig 1: ArhGEF12 is required for serum signaling to RhoA and for YAP1 activation. (A) MCF7 cells transduced with lentivirus encoding short hairpin (sh) control, shArhGEF11, or shArhGEF12 and treated with media for 30 min containing 0% or 10% serum. Fold-change in relative CTGF transcript levels (Top Graph). Immunoblot (IB) analysis of ArhGEF11, ArhGEF12 and GAPDH (bottom panels). (B) Immunoblot analysis measuring YAP1, snRNP70 and GAPDH protein levels in nuclear and cytosol fractions prepared from MCF7 cells described in A. (C) Ratios of RhoA-GTP and total RhoA levels were measured by G-LISA, Cytoskeleton, Inc. from lysates from MCF7 cells with shcontrol, shArhGEF11 or shArhGEF12. Cells were serum-starved for 24 h before treatment with 0% or 10% serum for 30 min. (D) MCF7 cells stably transduced with shRNA control were mixed with cells transduced with either shArhGEF11 or shArhGEF12 and then immunostained with antibodies against ArhGEF11 or ArhGEF12 in combination with phalloidin-CruzFluor 594. Dashed lines demarcate cell clusters with normal expression versus cells silenced for either GEF. (E) Brightfield images of MCF7 cells silenced for ArhGEF11 (Bottom Left) or ArhGEF12 (bottom Right). (F, G) Fold-change in CTGF transcript levels induced by LPA (1 µM) or the PAR2-A peptide (5 µM) in serum starved MCF7 cells stably transduced with either shControl (−) or shArhGEF11 (in G) or shArhGEF12 (in H). (H, I) fold-change in the induction of CTGF transcript by Gα12 (in J) or Gα13 (in K) in MCF7 cells transduced with shControl (−), shArhGEF11 or shArfGEF12. (J) Fold-change in CTGF transcript levels induced by LPA (1 µM) or Bradykinin (10 nM) in MCF7 cells transduced with shControl, shArhGEF11 (shAG11) or shArhGEF12 (shAG12). In I/J, *p-value of < 0.05 computed by student t-test.