Figure 3

HF induces Mmp13 expression in assay that does not inhibit TGFβ signaling or induce Smad 7. (A) HF and MAZ1319 but not MAZ1310 induce transcription of Mmp13 (A) in NMuMG cells after 8 hours of treatment, consistent with activities determined for HF analogs for Smad7 induction. Note that 0.01 μM HF treatment induces Mmp13 expression, but not Smad7 expression in this assay. All samples were normalized to β-Actin transcript levels. The fold induction of Mmp13 is calculated relative to DMSO vehicle treatment. (B) HF does not affect endogenous Phospho-Smad3 levels in cells at minimum concentration required to induce Mmp13 expression (0.01 μM). Cells were treated with the indicated HF concentrations or with DMSO (-) and were cultured in complete media for 8 hours. Cells were harvested after 8 hours and processed for SDS-PAGE analysis. Phospho-Smad3 was detected using a polyclonal Phospho-Smad3 antibody and Tubulin levels are shown as a loading control.