Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing - Fig 1

<p>(A), histologic findings in the patient’s kidney biopsy. Periodic acid–Schiff (PAS) staining (magnification 200X) shows segmental glomerulosclerosis and interstitial fibrosis with multiple foci of microcystic tubular dilation. (B), Electron microscopy images shows glomerular podocyte foot processes effacement (arrows) (magnification 20000X). (C), serum creatinine and urine protein/creatinine ratio of the patient over a period of a year.</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

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

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

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 model depicting the role of JunB in regulation of smooth muscle contractility in response to TGFβ1 signaling.

<p>TGFβ1 induces the expression of JunB as well as other markers of smooth muscle differentiation e.g. α-SMA, calponin and SM22α Additionally, TGFβ1 also promotes smooth muscle contraction via ROCK1-mediated regulation of actin polymerization and acto-myosin crossbridge cycling. JunB mediates this process by promoting the phosphorylation of cofilin, leading to stabilization of filamentous actin and also by regulating the phosphorylation and absolute levels of MLC20, the regulatory light chain of myosin, and its inhibitory phosphatase, MYPT1. Thus, activation of JunB is critical for the changes in contractility and generation of cytoskeletal tension observed upon the TGFβ1-stimulation of smooth muscle cells.</p

JunB levels are increased in BSMC in response to TGFβ1, and in an ex vivo model of rodent bladder distension.

<p>(<b>A</b>) BSMC were treated with TGFβ1 for the indicated times and assessed for JunB levels by immunoblotting. GAPDH is included as a loading control. (<b>B</b>) Immunofluorescence analysis of BSMC showing increased JunB nuclear localization upon TGFβ1 treatment for 24 h. (<b>C</b>) Sections from rat bladders distended ex vivo for 8 h (injured) were stained sequentially with anti-JunB and Cy3-conjugated species-specific secondary antibody. Increased nuclear fluorescent signal for both proteins was evident in the detrusor smooth muscle of stretch-injured specimens, but not of non-distended (control) bladders.</p

TGFβ1induces contractility in bladder smooth muscle cells (BSMC).

<p>(<b>A</b>) Human bladder smooth muscle cells were seeded in collagen gels and treated for 24 h with vehicle (Veh) or 2.5 ng/ml TGFβ1, after which the gels were released from the sides of the well and the resulting decrease in surface area monitored microscopically (top) and quantified (bottom). *p<0.05, t-test. The area of the gel under control conditions is set to 100%. (<b>B</b>) Whisker plot of results from traction force microscopy of BSMC showing an increase in cell traction forces exerted with TGFβ1 treatment. The contractile response, measured quantitatively as enhanced 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, Kruskal-Wallis test). The median value of traction and the interquartile range for both groups is shown. (<b>C</b>) BSMC were treated for 30 min with inhibitors targeting the PI3-kinase/Akt (PI3K-i, Akt-i) mitogen-activated protein kinases (MEK-i, p38-i, JNK-i) or Rho-kinase (ROCK-i), followed by treatment with vehicle (Control, upper panel of wells) or 2.5 ng/ml TGFβ1 (lower panel) for 24 h and were monitored for changes in gel contractility. Inhibition of signaling via the JNK and ROCK axes abrogated TGFβ1-induced gel contraction. Quantification of changes in gel surface area for the various inhibitors under conditions of TGFβ1 treatment is indicated. (<b>D</b>) A transcription factor ELISA was carried out to assess differences in DNA-binding activities of members of the AP-1 family of transcription factors, using nuclear extracts prepared from BSMC treated with 2.5 ng/ml TGFβ1 for 24 h, or control cells. Fold changes are expressed relative to control which is set to 100%.</p