Fig. 3.

MMP3 activity is necessary for E2-mediated cleavage of FasL. (A) U2OS-ERα cells were transfected with FASL-EGFP then treated with vehicle control (EtOH), 10 nM E2 and/or 1 µM MMP3 inhibitor (MMP3I) for 24 hours. Cells were analyzed by flow cytometry and the percent change in EGFP compared to EtOH treated cells (set at 100%) is graphed. Four biological replicates are averaged. (B) U2OS-ERα cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). 48 hours after transfection cells were treated for 24 hours with vehicle control (C) or 10 nM E2. Cells were lysed and total cellular protein was immunoblotted for FasL, MMP3 and actin. (C) U2OS-ERα cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). 48 hours after transfection cells were lysed, and cDNA was analyzed by qPCR for MMP3, MMP7 and ADAM10. (D) U2OS-ERα cells were transfected with either an siRNA directed at luciferase (siLUC) or MMP3 (siMMP3). 48 hours after transfection cells were treated for 24 hours with vehicle control (C) or 10 nM E2. Cells were lysed and total cellular protein was immunoblotted for ADAM10, MMP7 and actin. Human osteoblasts (hOB) and MCF7 cells were used as controls. * = p-value <0.05.