Figure 3

Eplin-α expression is controlled by mutant actins and MAL/MRTF transcriptional coactivators. (A) Cotransfection of NIH 3T3 cells with the indicated Eplin-α reporter and actin wildtype (wt), non-polymerisable mutant actin R62D, and F-actin stabilising mutant actin G15S [16, 17]. Following cytochalasin treatment, the relative Luciferase activity was determined as before. (B) Transient expression of constitutively active MAL ΔN or MAL affect the Eplin-α mRNA level. Cells were transiently infected with the indicated pLPCX-derived retrovirus [19]. Two days later, the total mRNA was isolated and analysed for Eplin-α mRNA by quantitative RT-PCR as before. (C) Effect of dominant negative MAL ΔNΔB, MAL ΔNΔC, or double knockdown of MRTFs, on serum-stimulated Eplin-α expression. Infection was done with derivatives of either pLPCX or pSUPER-Retro (pSR, control), generating a shRNA targeting both MAL/MRTF-A and MRTF-B [19]. Two days after infection, cells were stimulated with serum if indicated (FCS, 15%, 90 min), and the relative Eplin-α mRNA induction was quantified. (D) Effect of constitutively active MAL ΔN and MAL, or double knockdown of MRTFs, on Eplin-α expression in EpRas epithelial cells. Shown is the average mRNA amount of EpRas cells stably infected with the indicated MAL constructs, compared to the mock-infected control cells (pLPCX and pSR control, respectively). Error bars, SEM (n = 3). Asterisk, significant activation; double asterisk, significant repression (p < 0.01, unpaired student's t-test).