(A) TPA-induction of MRTF target genes. Cells were stimulated with 100 ng/ml TPA, and MRTF-A target gene pre-mRNA at 0.25, 0.5, 1, 2 and 3 hr analysed by qPCR, normalised to GAPDH and expressed relative to uninduced conditions. Data are mean ± half-range, n = 2. (B–D). Cells expressing Flag-MRTF-A or derivatives were pretreated with the indicated inhibitors for 20 min (U0126, 10 μM; Gö6976, 2 μM; Gö6983, 6 μM), then treated with 100 ng/ml TPA for 20', with serum stimulation for 20' as indicated. MRTF-A subcellular localisation was assessed by immunofluorescence. Data are mean ± half-range, n = 2. (B) TPA-induces MRTF-A nuclear accumulation (p<0.01, two-way ANOVA, Bonferroni post-test) but not in presence of inhibitors. (C) Cells as in (B) were stimulated with 15% FCS. Data are mean ± half-range, n = 2. Serum-induced MRTF-A nuclear accumulation was inhibited by pre-treatment with TPA (p<0.01), TPA+U0126 (p<0.01), and TPA+Gö6976 (p<0.05), but not TPA+Gö6983 (ns); two-way ANOVA with Bonferroni post-test. (Part B shows only the t = 0 points from this experiment for clarity). (D) Cells expressing Flag-MRTF-A, Flag-MRTF-A STS/A (S544A/T545A/S549A), or MRTF-A(2–204)-PK, together with Raf-ER as indicated, were pre-treated for 20' with 100 ng/ml TPA or 1 μM 4-hydroxytamoxifen (4OHT) then stimulated with 15% FCS for 20 min. Data are from two (WT and STS/A) or four independent experiments ((2–204)-PK). Only pre-treatment with TPA significantly blocked serum-induced nuclear accumulation (Predominantly nuclear MRTF-A cells: WT and STS/A, p<0.01; 2–204 PK, p<0.05; one-way ANOVA, Bonferroni multiple comparison test). (E) TPA inhibition of F-actin assembly in NIH3T3 cells is PKC-dependent but does not require ERK or conventional PKC. Cells were pretreated with the indicated inhibitors for 20 min, treated with 100 ng/ml TPA for 20 min, then stimulated with 15% FCS for 20 min, as in (C). After fixation F- and G-actin were quantified using fluorescently labelled phalloidin and DNase I, respectively, with F:G ratio in unstimulated cells set arbitrarily to unity. Plots are mean ± SEM, n = 3, with significance estimated by one-way ANOVA (*p<0.05; **p<0.01; ***p<0.001). (F) TPA inhibition of serum-induced RhoA activation and MLC2 phosphorylation is PKC-dependent but does not require ERK or conventional PKC. Lysates from cells treated as in panel (E) were used in a RhoA.GTP pull-down assay with GST-rhotekin as the affinity matrix (top); or analysed for MLC2 S19 phosphorylation by immunoblotting (bottom).