Figure 1.
Combinations of EGCG and luteolin synergistically inhibit TGF-β-induced fibronectin expression and reduce fibroblast matrix contraction.
(A–E) WPMY-1 and HPS-19I cells were treated with or without 5 ng/ml TGF-β in the presence of EGCG and/or luteolin at the indicated concentrations for 24 hours. Cell lysates were analyzed by western blot and densitometry was used to determine reduction of fibronectin expression. (A) Dose response data are expressed as mean ± S.E.M.; n = 3. (B) WPMY-1 synergy was determined using CompuSyn software, synergy = CI<1; n = 3. (C) Representative western blot of synergistic combinations of EGCG and luteolin with corresponding single agent doses. (D) HPS-19I synergy was determined using CompuSyn software, synergy = CI<1; n = 3. (E) WPMY-1 cells were treated with or without 5 ng/ml TGF-β in the presence of EGCG and/or luteolin at the indicated concentrations for 4 days post seeding. Data are shown as percent contracted area; n = 3.
Figure 2.
EGCG and luteolin do not synergistically reduce WPMY-1 cell proliferation and viability.
WPMY-1 cells were treated with EGCG and/or luteolin at the indicated concentrations for 24 hours in 1% FBS DMEM (A) or 96 hours in 5% FBS DMEM (B). Proliferation (indicated by bars; left y-axis) and metabolically viable cells (indicated by line; right y-axis) are shown as mean percent confluency or viability ± S.E.M. normalized to TGF-β; 1%: n = 2, 5%: n = 3.
Figure 3.
EGCG may block fibronectin induction through inhibition of ERK.
(A) WPMY-1 cells were treated with or without 5 ng/ml TGF-β for 24 hours and PI-3K inhibitor LY294002 or ERK inhibitor U0126, EGCG or luteolin at the indicated concentrations. Cell lysates were analyzed by western blot. (B) WPMY-1 cells were treated with or without 5 ng/ml TGF-β in the presence of 20 µM luteolin or 40 µM EGCG for 1 hour prior to nuclear fractionation. Western blot was used to analyze 5 µg of protein per sample from each fraction.
Figure 4.
Fibronectin induction is not dependent on lipid raft integrity.
(A) WPMY-1 cells were treated with or without 5 ng/ml TGF-β in the presence of 10 or 40 µM EGCG or 5 or 20 µM luteolin for 24 hours. (B) Cells were treated with the indicated concentrations of mβCD for 30 minutes. (A and B) Cells were stained with 1 µM DiIC16 (red) and DAPI (blue). Representative images are shown. (C) Cells were treated with 1.2–15 mM mβCD for 30 minutes, washed, then treated overnight with TGF-β. Indicated proteins were analyzed by western blot.
Figure 5.
Rho activity is required for WPMY-1 fibronectin induction.
(A) qRT-PCR was performed, following RNA isolation and cDNA synthesis, on WPMY-1 cells under normal culture conditions. Expression of the indicated genes was analyzed; n = 2. (B) WPMY-1 cells were pre-treated with the Rho A/B/C inhibitor C3 Transferase at 1.2–10 µM for 8 hours prior to addition of 5 ng/ml TGF-β for 24 hours. Cell lysates were analyzed by western blot.
Figure 6.
EGCG and luteolin reduce RhoA activation and reverse TGF-β-induced fibronectin expression.
(A) WPMY-1 cells were pre-treated 8 hours with 20 µM GGTI-2133, 20 µM EGCG or 20 µM luteolin in the presence or absence of 10 µM or 40 µM geranylgeranylpyrophosphate (GGPP) prior to co-treatment with TGF-β for an additional 24 hours. Western blot was used to determine levels of the indicated proteins. (B) WPMY-1 cells were pre-treated 30 minutes in 1% FBS DMEM with EGCG (E) or luteolin (L) prior to addition of TGF-β for an additional 15 minutes. Rhotekin-RBD beads were used to pulldown active RhoA. (C) WPMY-1 cells were pulsed with TGF-β for 24 hours, washed with PBS, then chased with 40 µM EGCG or luteolin with or without TGF-β for an additional 24 hours.