WFA prevents myofibroblast transformation and collagen gel contraction in RbTCF cells.

<p>Immunohistochemical analysis of vimentin (<i>green</i>) and α-SMA (<i>red</i>) in RbTCF cells treated with TGF-β1 (2 ng/ml) for 3 days in presence and absence of WFA. (A) TGF-β1 treatment induces formation of spheroid-like agglomerates (upper panels, 10× magnification) with sprouting extensions positive for vimentin (<i>green</i>) and α-SMA (<i>red</i>) (lower panels, 30× magnification). (B–C) WFA causes a dose-dependent downregulation of both vimentin (<i>green</i>) and α-SMA (<i>red</i>) expression. Upon treatment with 500 nM WFA, cells form spheroids poorly (C, insert panels) and those that do form spheroids have shorter and thinner extensions (C, <i>arrowheads</i>). Scale bars: 70 µm at 10× magnification; 30 µm at 30× magnification. (D) Western blot analysis of soluble vimentin and α-SMA expression in RbTCF cells (left panel) and RbCF cells (right panel). (E) Densitometric quantification of vimentin (<i>black and orange</i>) and α-SMA (<i>grey and blue</i>) in RbTCF cells and RbCF cells, respectively, normalized to GAPDH using ImageJ software. (F) Representative images of polymerized collagen gels containing RbTCF cells (n = 6 gels per treatment group) treated with TGF-β1 (2 ng/ml) in presence and absence of different doses of WFA for 3 days (10× magnification). (G) Quantification of percentage of gel contraction normalized to the initial size of the gel. Data are the mean of 2 independent experiments; Dotted circles represent the well's area. *P<0.05 TGF-β vs WFA treated cells.</p

WFA distribution in anterior segment tissues.

<p>WFA distribution in anterior segment tissues.</p

Identification of RbTCF cells <i>in vivo</i> and <i>in vitro</i>.

<p>Immunohistochemistry analysis of cell marker vimentin in rabbit Tenon's capsule tissue (B, <i>red</i>) and in isolated RbTCFs (A, <i>green</i>). Scale bars: 100 µm <i>in vitro</i> (A), 50 µm <i>in vivo</i> (B).</p

Bleb appearance in representative rabbit eyes subjected to GFS.

<p>(A) WFA treated eyes (top panels a–c; high dose) and vehicle treated eyes (bottom panels d–f) at weeks 1, 2 and 3 post-surgery. The arrows indicate the presence of the bleb and arrowheads point to the cannula. Note the increased vascularization at two weeks post surgery (e). (B) Kaplan-Meier bleb survival plot of rabbit eyes treated with vehicle (solid line), low dose WFA (dotted line) and high dose WFA (dashed line).</p

WFA downregulates soluble vimentin and affects the skp2-p27^kip1 anti-fibrotic pathway in an experimental glaucoma filtration surgery model in rabbit.

<p>(A) Immunohistochemical analysis of vimentin expression (<i>red</i>) in Tenon's capsule tissues at surgical sites 28 days post-injury (30× magnification). Note the change in cell shape (<i>arrowhead</i>) in the injured sample (Veh) with abundant cytoplasmic vimentin staining (<i>red</i>) as compared to uninjured sample (Cont) and the downregulation and fragmentation of vimentin-IFs in the high dose WFA-treated sample. (B and E) Western blot analysis of representative tissues from Tenon's capsule tissues extracts at 28 days post-injury showing expression levels of vimentin, α-SMA, skp2 and p27^kip1 in low-salt buffer extracts (soluble fraction). Blots were re-probed consecutively with antibodies. (C–D and F–G) Densitometric quantification of soluble vimentin (<i>black</i>), α-SMA (<i>grey</i>), skp2 (<i>shaded grey</i>) and p27^kip1 (<i>shaded black</i>) normalized to GAPDH, using ImageJ software. Scale bars: 50 µm at 30× magnification. *P = 0.0012 Cont vs Veh; **P = 0.0005 Veh vs WFA high dose; ***P = 0.0069 Veh vs WFA low dose (<i>Panel C</i>). *P = 0.0001 Cont vs Veh; **P = 0.0001 Veh vs WFA high dose; ***P = 0.0001 Veh vs WFA low dose (<i>Panel D</i>). *P = 0.0001 Cont vs Veh; **P = 0.00005 Veh vs WFA high dose; ***P = 0.0012 Veh vs WFA low dose (<i>Panel F</i>). *P = 0.0357 Cont vs Veh; **P = 0.0005 Veh vs WFA high dose; ***P = 0.0012 Veh vs WFA low dose (<i>Panel G</i>).</p

WFA inhibits cell migration in RbTCF cells.

<p>Scratch wound assay <i>in vitro</i>. (A) Representative phase-contrast images of RbTCF cells immediately after a cross-stripe scrape injury on a confluent cell layer (0 h), 24 h post-wounding (Veh), and after treatment with different concentrations of WFA. Mitomycin-C (MMC; 15 µM) was used as positive control. (B) Arbitrary values of wound gap at 24 h normalized to size of initial wound gap and calculated from two independent experiments (n = 3 for each treatment group). *P<0.05 vehicle vs drug treated; ns = non-significant. (C) Quantification of total numbers of DAPI nuclei per wound area at 24 h post-scratch injury (**P<0.0001 vehicle vs drug treated) and Ki-67 positive cells (blue bars; ^#P<0.0001 vehicle vs drug treated). (D) Fraction of Ki67 positive nuclei in the wound area as a percentage of DAPI nuclei at 24 h post-scratch injury. *** P<0.0001 vehicle vs drug treated.</p

WFA's cell cycle effects are regulated by the skp2-p27^kip1 pathway in RbTCF cells.

<p>(A) Representative images of RbTCFs stimulated to enter the cell cycle after 48 h serum starvation. Cells were stained with vimentin (<i>green</i>) and phalloidin (<i>red</i>) at 24 h post-WFA treatment. WFA's low concentration cell cycle activity does not affect the vimentin-IF network (<i>green</i>). Vimentin-IFs become affected only at higher concentrations (1 µM, <i>arrow heads</i>) and leads to dismantling of the actin fibers (<i>red</i>) that are observed at the periphery of cytoplasm (<i>arrows</i>). (B) Western blot analysis of skp2 and p27^kip1 expression in RbTCF cells at 24 h post-WFA treatment. C) Densitometric quantification of skp2 (<i>black</i>) and p27^kip1 (<i>grey</i>) normalized to β-actin, using ImageJ software. Scale bars: 30 µm at 30× magnification. * P<0.05 WFA vs control.</p

WFA potently arrests RbTCFs in G₀/G₁ phase.

<p># mean of two experiments.</p