Fig. 2. TGFβ1 and Smad3 promote vimentin reporter gene activity in C2C12 cells via the tandem AP-1 elements.

(A) Schematic diagram of the human vimentin promoter depicting its various cis-elements. The negative numbers (marked by a vertical arrow) indicate the 5′-end of different vimentin promoter constructs, which extend to +72 at the 3′-end, fused to the reporter gene, CAT. The transcriptional start site is noted by a horizontal arrow at +1. (B) Various vimentin promoter CAT constructs (-775CAT, -725CAT, -353CAT, -319CAT and -261CAT) were transiently transfected into C2C12 cells. Myoblast cells (MB) maintained in growth medium were harvested 48 h after transfection whereas myotubes (MT) were harvested 72 h after transfection and transfer to differentiation medium. Reporter gene activity was normalized to co-transfected beta-galactosidase as an internal control. Results are the average of three separate experiments performed in triplicate and bars represent the standard error. (C) Vimentin promoter construct -775CAT was transiently transfected into C2C12 cells with or without Smad3 expression plasmids as indicated. Cells were grown in either GM or DM without or with TGFβ1 as indicated. Reporter gene activity was measured as above. (D) The -775CAT or -775mAP-1CAT construct containing mutant tandem AP-1 sites was transfected into C2C12 myoblasts with either the empty vector or vector containing Smad3 without or with TGFβ1 as indicated. See Fig. 4B for sequence of the AP-1 and mAP-1 region. Reporter gene activity was measured as above. (E) Schematic structure of Smad3 indicating its deletion mutants. (F) The -775CAT construct was transiently transfected into C2C12 myoblasts with either the empty vector or vector expressing Smad3 or mutants thereof (Smad3NL or Smad3C) as indicated. Reporter gene activity was measured as above.