JCS013557 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Active RhoA enhances the interaction between mDia1 and POPX2. (A) Flag-mDia-FL was co-transfected with GST-POPX2 and pXJ-HA vector (lane 1), HA-RhoV14 (lane 2) or HA-RhoN19 (lane 3) into COS7 cells. Cell lysates were incubated with glutathione beads. The protein bound was eluted and analyzed by SDS-PAGE and western blotting using anti-Flag antibodies to detect co-purification of Flag-mDia molecules. (B) Active RhoA does not enhance the interaction between POPX2 and mDia in ‘open’ conformation. Flag-mDiaΔC was co-transfected with GST-POPX2 with (lane 2) or without (lane 1) HA-RhoAV14. The GST-pulldown was analyzed by SDS-PAGE and western analysis to detect the co-purification of Flag-mDia-ΔC.

• Supplemental Figure S2 -

  Fig. S2. Different isoforms of mDia can interact with POPX1 and POPX2. (A) mDia2 interacts with POPX2. Different Flag-tagged mDia2 constructs were co-transfected with GST-POPX2 into COS7 cells. The GST pulldown complex was analyzed by SDS-PAGE and western analysis to detect co-precipitation with GST-POPX2. Lane 1, Flag-mDia2-FL + GST-POPX2; 2, Flag-mDia2-DN + GST-POPX2; 3, Flag-mDia2-ΔGBD + GST-POPX2; 4, Flag-mDia2-ΔN1 (FH1 to stop) + GST-POPX2. (B) POPX1 interacts with activated mDia1 and mDia2. Flag-tagged mDia1 and mDia2 constructs were co-transfected with GST-POPX1. The GST pulldown complex was analyzed by SDS-PAGE and western analysis to detect co-precipitation with GST-POPX1. Lane 1, Flag-mDia1-FL + GST-POPX1; 2, Flag-mDia1-ΔGBD + GST-POPX1; 3, Flag-mDia2-FL + GST-POPX1; 4, Flag-mDia2-ΔGBD + GST-POPX1.

• Supplemental Figure S3 -

  Fig. S3. POPX2 siRNA knockdown exerts a positive effect on transcription from SRE. Different constructs and siRNAs were transfected into COS7 cells and the luciferase activity from the reporter was measured as described in the Material and Methods. (A) The transfections were: lane 1, SRE control; 2, RhoAV14; 3, POPX2 siRNA1. (B) The transfections were: lane 1, SRE control; 2, RhoAV14; 3, RhoV14 + POPX2 siRNA1; 4, RhoAV14 + negative siRNA control.

• Supplemental Figure S4 -

  Fig. S4. POPX2 blocks the effect of dominant-negative mDia. Flag-mDia-DN was transfected either with or without various POPX2 constructs into HeLa cells. The cells were fixed and immunostained with anti-Flag antibodies and rhodamine-coupled phalloidin. Cells transfected with just mDia-DN alone showed loss of stress fibres. Interaction between POPX2 and mDia-DN was required to block the mDia-DN phenotype. The phenotype was scored by analyzing at least 100 cells per transfection. Each transfection was repeated three times. Lane 1, HeLa transfected with GFP vector; 2, mDia-DN; 3, mDia-DN + POPX2; 4, mDia-DN + POPX2m; 5, mDia-DN + POPX2N; 6, mDia-DN + PPc; 7, mDia-DN + PPm.

• Supplemental Figure S5 -

  Fig. S5. ROK and mDia induce the formation of different types of actin structures. HeLa cells were transfected with different cDNA constructs encoding GFP, Flag-tagged wild-type ROK and Flag-mDia-ΔGBD. The cells were fixed and immunostained with anti-Flag antibodies and rhodamine-coupled phalloidin. A duplicate set was treated with 10 µM 27632 (ROK inhibitor) for 1 hour before fixing. Cells transfected with mDia-ΔGBD showed long and thin actin filaments. These filaments were not disrupted by the use of ROK inhibitor. Arrows represent transfected cells. Star in lower-right panel represents an untransfected cell with loss of stress fibres after treatment of ROK inhibitor. Scale bar: 10 µm.