Urothelial cell attachment and proliferation are modulated by silk biomaterial morphology and selective ECM coatings.

<p>[A] AlamarBlue analysis of the extent of relative urothelial cell attachment and proliferation on uncoated or ECM-coated (collagen types I or IV or fibronectin) silk groups following 1 and 7 d of culture, respectively. FU =  Fluorescent units, mean ± SD per data point. (*)  =  <i>p</i>≤0.05, in comparison to levels observed on respective 1 d counterparts. [B] Photomicrographs of urothelial cell-seeded constructs (H&E-stained sections) following 7 d as described in [A]. Scale bar  =  100 µm; inset, scale bar  =  50 µm. [C] Photomicrographs of pan-cytokeratin protein expression in urothelial cells cultured for 9 d on control and fibronectin-coated groups. Immunofluorescence of pan-cytokeratins (Cy3 fluorophore, red). DAPI nuclear counterstain (blue). Scale bars  =  100 µm; magnified inset, scale bars  =  50 µm. For [B,C], SC denotes scaffolds.</p

Stiffness dynamics in HBSM cells subjected to a transient stretch or a transient compression.

<p>In response to a transient tensile stretch, the elastic modulus G' dropped acutely to 50–60% of its prestretch value, then recovered slowly. The rate and the extent of this fluidization-resolidification is unaffected by the addition of LY294002, Triciribine, SB2035580, PD098059, Y27632, or Latrunculin-A (p≫0.05, Kruskal-Wallis one-way analysis). When we applied to the cell a transient compression instead of a transient stretch, G' was unchanged. All results are reported as medianSE (n = 244–709 beads).</p

Structural morphology of silk scaffold groups and characterization of ECM coatings.

<p>SEM photomicrographs of cross-sectional and top views (inset) of silk scaffold configurations. Scale bars  =  300 µm; inset, scale bars  =  200 µm. Photomicrographs of H&E-stained cross-sections of unseeded scaffold groups and immunofluorescence of various ECM coatings (collagen types I or IV or fibronectin) on matrices prior to cell seeding with similar orientation (Cy3 fluorophore, red). Scale bars  =  100 µm. SC denotes scaffolds. Arrows denote intercalation of ECM coatings within scaffold pores.</p

Group 2 silk scaffold architecture coupled with fibronectin coatings maximizes pluripotent stem cell attachment.

<p>[<b>A</b>] Photomicrographs of phase contrast images of naïve ESC and iPS cells prior to scaffold seeding. Immunofluorescence of the pluripotent marker, OCT4, in ESC and iPS cells (Cy3 fluorophore, red). DAPI nuclear counterstain (blue). Scale bars for phase and immunofluorescence images  =  100 µm. [<b>B, C</b>] AlamarBlue analysis of the extent of relative ESC [B] and iPS cell [C] attachment on uncoated or ECM-coated (collagen types I or IV or fibronectin) silk groups following 1 d of culture. FU =  Fluorescent units, mean ± SD per data point. (*)  =  <i>p</i>≤0.05, in comparison to levels observed on respective uncoated controls.</p

A major component of fluidization is F-actin disassembly: staining results.

<p>Within each well, F-actin levels in stretched HBSM cells (inside the indenter footprint) were compared to those in unstretched cells (outside the indenter footprint). (A),(C), F-actin filaments rapidly dissociated immediately after stretch. (B),(D), the F-actin levels recovered to baseline 300 seconds after stretch. (E),(F), from those images, we quantified intensities. Within each dish, we normalized average F-actin intensities in stretched cells to those in unstretched cells (6 dishes in total for each condition) and pooled the values together. Average F-actin intensity in stretched cells was 50% lower than that in unstretched cells immediately after stretch (Figure 3E; p<0.05, two-tailed unpaired Student's t-test), but recovered to prestretched values 300s after stretch (Figure 3F). Data are reported as meanSE (n = 14–29 cells).</p

Traction force dynamics in HBSM cells subjected to a transient stretch.

<p>(A) Cell traction map before stretch. (B) Traction map immediately after a 10% homogeneous biaxial tensile stretch of a 4s duration. The cell tractions are markedly ablated. (C) Traction map measured at 300s following stress cessation. Tractions have largely recovered to the baseline value in (A). (D) Contractile moment computed from the traction maps. After transient stretch, the contractile moment decreased by 70% and then recovered to 60% of baseline value in 300s. Data are reported as meanSE (n = 9 cells).</p

SMC and urothelial populations in RA-treated pluripotent stem cells cultured on silk scaffolds.

<p>[<b>A, B</b>] Photomicrographs of SMC contractile protein expression (α-actin and SM22α) and urothelial-associated markers [pan-cytokeratins (CK) and pan-uroplakins (UP)] in ESC [A] and iPS cells [B] cultured on fibronectin-coated Group 2 matrices for 14 d in the presence of RA. (Cy3 fluorophore, red). DAPI nuclear counterstain (blue). Scale bars  =  100 µm; magnified inset, scale bars  =  50 µm. SC denotes scaffolds in all panels.</p

JunB regulates actin polymerization.

<p>(<b>A</b>) JunB silencing in BSMC reduces phospho-cofilin levels under basal and TGFβ1-stimulated conditions, without affecting total cofilin levels. *p<0.05; **p<0.005. Representative immunoblots are indicated in (<b>B</b>). (<b>C</b>) Filamentous (F) and globular (G) actin fractions were purified as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053430#s2" target="_blank">Methods</a>, from pBSMC under vehicle or TGFβ1-treated conditions, following treatment with non-targeting or JunB siRNA. The relative levels of F- and G-actin were subsequently assessed by immunoblotting. Quantification of immunoblot signals from three independent experiments is shown. *p<0.05. Representative immunoblots are indicated in (<b>D</b>).</p

JunB silencing attenuates TGFβ1-induced changes in cell contractility and cytoskeletal tension, but not induction of markers of smooth muscle differentiation.

<p>(<b>A</b>) BSMC were nucleofected with non-targeting control siRNA or with siRNA against JunB (0.1 µM and 1 µM) and assessed for JunB protein by immunoblotting (left panel, top). Effective knockdown of JunB was observed, with no change in c-Jun levels, demonstrating specificity of the siRNA used. Proliferating cell nuclear antigen (PCNA) expression was used as a loading control. 1 µM JunB siRNA reduced the levels of JunB mRNA by >80%, relative to non-targeting control siRNA, as assessed by semi-quantitative real-time PCR (right panel) (<b>B</b>) Reduction in JunB protein levels by siRNA in BSMC under basal and TGFβ1-stimulated conditions, demonstrated by immunoblotting. JunB levels were normalized to their respective GAPDH levels and expressed as percentage change relative to cells transfected with control siRNA and not subjected to TGFβ1 treatment. A representative immunoblot and its corresponding quantitation are shown. (<b>C</b>) TGFβ1-mediated induction of α-smooth muscle actin (α-SMA) calponin and SM22α, markers of smooth muscle differentiation, was unaffected by silencing of JunB, as shown by immunoblotting (left). Quantification of immunoblots is shown in the graph (right). Gel contraction assays (<b>D</b>) revealed that JunB knockdown significantly reduced both basal and TGFβ1-induced changes in cellular contractility. *p<0.05, t-test (<b>E</b>) Inhibition of JunB inhibits basal and TGFβ1-induced contraction. This inhibition of contraction, measured quantitatively as a reduction of traction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053430#s2" target="_blank">Methods</a>) was statistically significant (*p<0.05, comparing siCtrl+ TGFβ1 or siJunB-TGFβ1 with siCtrl-TGFβ1; ∧p<0.05 comparing siCtrl+ TGFβ1 with siJunB+ TGFβ1 Kruskal-Wallis test). The median value of traction and the interquartile range across all tested groups is shown.</p

A major component of fluidization is F-actin disassembly: molecular blotting.

<p>(A) A transient stretch did not affect phosphotyrosine protein phosphorylation profiles. Positive control: PDGF and HB-EGF treatment for 5 minutes. (B) In contrast, a transient stretch acutely ablated F-actin levels at short times (S'0 – immediately after stretch). These levels recovered to baseline at longer times (S'2.5–2.5 minutes after stretch; S'5–5 minutes after stretch). Positive control (PC - 1µM phalliodin), negative control (NC - 10µM cytochalasin).</p