Journal: PLoS ONE

Article Title: Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

doi: 10.1371/journal.pone.0012626

Figure Lengend Snippet: The phenotype of mutant (b) and morphant (c) embryos were compared to wildtype zebrafish embryos at 120 hpf (a). The mutant and morphant embryos had several aberrant features including edema around the yolk, pericardial effusion, smaller eyes, an arched back, jaw malformation and an absent swim bladder. Control Wt1b-GFP transgenic zebrafish (d–f, d′–f′) and those treated with cofilin-1 morpholinos (g–i, g′–i′) were injected with 70 kDa rhodamine labeled dextrans at 72 hpf and their pronephros imaged 48 hours later. The rhodamine labeled dextrans remained within the circulatory system of the Wt1b-GFP zebrafish with no labeling within the Wt1b-GFP pronephros tubules (d–f, d′–f′). In contrast, in the Wt1b-GFP transgenic morphant embryos (g–i, g′–i′), the vasculature was labeled by the rhodamine labeled dextran, but labeling was also observed within the pronephros tubules (magnification = 180×). (j–j″) XZ cross section of the collected optical stacks clearly demonstrated the intracellular localization of rhodamine labeled dextrans within the pronephros tubules (yellow). (k) Functional studies demonstrated loss of pronephros function in cofilin-1 knockdown zebrafish embryos. Images of intravascular fluorescence were captured in retinal blood vessels and monitored in individual fish over 24 hours in control, cofilin-1 mutant zebrafish and zebrafish treated with 100µM cofilin-1 morpholinos. At forty-eight hours post injection of FITC-labeled 70 kDa dextrans into the vascular system, the fluorescence intensity in the pupil of the zebrafish increased in wildtype embryos, but decreased in zebrafish treated with 100 µM cofilin-1 morpholinos and cofilin-1 mutants. (l) TEM analysis of podocytes in control, cofilin-1 mutant and cofilin-1 morpholino treated zebrafish demonstrated severe podocyte effacement in cofilin-1 knockdown embryos. While wildtype (upper left) and control morpholino injected (lower left) zebrafish embryos have normal foot processes and slit diaphragm formation, the cofilin-1 mutants (upper right) as well as the cofilin-1 morpholino injected fish (lower right) display foot process fusion and foot process effacement over large areas (size bar: 500 nm).

Article Snippet: Western blots were probed with primary antibodies, p-cofilin (1∶1000) (Santa Cruz) or cofilin-1 (1∶5000) (Cytoskeleton) followed by the HRP labeled goat anti-rabbit secondary antibody (1∶10,000) (Santa Cruz).

Techniques: Mutagenesis, Control, Transgenic Assay, Injection, Labeling, Functional Assay, Knockdown, Fluorescence

Journal: PLoS ONE

Article Title: Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

doi: 10.1371/journal.pone.0012626

Figure Lengend Snippet: (A) Using a primary rabbit antibody to cofilin, a diffuse distribution of cofilin throughout the cytoplasm was observed in both murine and human control podocytes (2A, red). In wildtype murine and normal human glomeruli, expression of cofilin colocalized with synaptopodin, a podocyte specific marker (2A, merge white arrows, insert enlarged 10×). (B) Analysis of RNA lysates of conditionally immortalized murine and human podocytes demonstrated cofilin-1 is the dominant isoform compared to cofilin-2 and ADF expression levels. The concentration of the cofilin-1 message is approximately 10 times higher than cofilin-2 and 3 times higher than ADF in murine podocytes and approximately 10 times higher than ADF in human podocytes, whereas cofilin-2 mRNA is barely detectable in human podocytes. Murine and human podocyte ADF/cofilin isoform expression levels did not significantly differ between one and ten days of differentiation.

Article Snippet: Western blots were probed with primary antibodies, p-cofilin (1∶1000) (Santa Cruz) or cofilin-1 (1∶5000) (Cytoskeleton) followed by the HRP labeled goat anti-rabbit secondary antibody (1∶10,000) (Santa Cruz).

Techniques: Control, Expressing, Marker, Concentration Assay

Journal: PLoS ONE

Article Title: Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

doi: 10.1371/journal.pone.0012626

Figure Lengend Snippet: (A) Western blot analysis using the p-cofilin specific antibody demonstrated cofilin-1 regulation through serine 3 phosphorylation in murine and human podocytes treated with PMA and TGF-β. Phosphorylation profiles in response to 8 hours of treatment with TGF-β demonstrated a very strong increase in cofilin-1 phosphorylation resulting in cofilin-1 inactivation in murine and human podocytes. In contrast treatment with PMA led to dephosphorylation and activation of cofilin-1 in murine and human podocytes. GAPDH was probed as a protein loading control. (B) Densitometric summary of p-cofilin/GAPDH ratios of three independent experiments.

Article Snippet: Western blots were probed with primary antibodies, p-cofilin (1∶1000) (Santa Cruz) or cofilin-1 (1∶5000) (Cytoskeleton) followed by the HRP labeled goat anti-rabbit secondary antibody (1∶10,000) (Santa Cruz).

Techniques: Western Blot, Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control

Journal: PLoS ONE

Article Title: Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

doi: 10.1371/journal.pone.0012626

Figure Lengend Snippet: (A) Western blot analysis of control and cofilin-1 siRNA transfected cell extracts demonstrated cofilin-1 protein expression was silenced by siRNA transfection. (B) Both control and cofilin-1 siRNA cells treated with cytochalasin were observed for recovery at 0, 1, 2 and 8 hours post treatment. At 0 and 1 hour time points, no discernible difference was observed in the control and cofilin-1 siRNA treated cells. By 2 hours post treatment, the cytochalasin effects on the cofilin-1 siRNA treated cells was diminished compared to the control siRNA cells. By 8 hours a significant difference in cell shape, size and cell spread was observed in the cofilin 1 siRNA treated cells compared to control cells. (C) Actin polymerization assay demonstrated an increased accumulation of actin fibers in podocytes in the absence of cofilin-1 (red squares) compared to control silenced podocytes (green triangles). Pyrene Actin in the absence of cell lysates serves as background control (black circles). (D) Control and cofilin-1 siRNA treated podocytes grown to confluence were wounded (0 hours) and their recovery examined 24 hours later. Control siRNA podocytes migrated to fill in the wound region at a significantly faster rate than podocytes treated with cofilin-1 siRNA.

Article Snippet: Western blots were probed with primary antibodies, p-cofilin (1∶1000) (Santa Cruz) or cofilin-1 (1∶5000) (Cytoskeleton) followed by the HRP labeled goat anti-rabbit secondary antibody (1∶10,000) (Santa Cruz).

Techniques: Western Blot, Control, Transfection, Expressing, Polymerization Assay

Journal: PLoS ONE

Article Title: Cofilin-1 Inactivation Leads to Proteinuria – Studies in Zebrafish, Mice and Humans

doi: 10.1371/journal.pone.0012626

Figure Lengend Snippet: Glomeruli recovered from patients with glomerular diseases, FSGS, MCD and MGN, were probed with the rabbit primary antibody to p-cofilin and goat primary antibody to podocalyxin (PDX) and visualized using an Alexa488 labelled antibody raised against goat (green) and a Cy3-labelled secondary antibody against rabbit (red). Podocytes in these diseased glomerular tissues demonstrated an increased incidence of phosphorylated cofilin-1 in their nuclei (merged images ±DAPI). In contrast p-cofilin of normal control patients revealed no glomerular staining indicating that the cofilin-1 expressed in the glomerulus under normal conditions is active and under disease-conditions is inactivated.

Article Snippet: Western blots were probed with primary antibodies, p-cofilin (1∶1000) (Santa Cruz) or cofilin-1 (1∶5000) (Cytoskeleton) followed by the HRP labeled goat anti-rabbit secondary antibody (1∶10,000) (Santa Cruz).

Techniques: Control, Staining