Knockdown of ACTN4 limits the number of focal adhesions.

<p>(A) WT ACTN4 and ACTN4 KD murine lung fibroblasts were stained for vinculin aggregates as indicative of focal adhesions. The number of focal adhesion sites residing within cell body and cellular edge were calculated by using ImageJ software for at least 30 individual cells. The ratio of focal adhesion sites between cell body and edge was also calculated. Images were taken randomly. Quantification was measured from at least 30 individual cells (± the standard deviation) chosen randomly. Shown are representative of at least independent experiments. Bar  = 10 µm. (B) An inverted centrifugation focal adhesion assay was performed (see “material and method” for details). Data are mean ± s.e.m., measured from three independent experiments. (C) Immunoblotting shows that the vinculin level was similar in WT and KD cells. Blot is representative of three independent assessments.</p

Knockdown of ACTN4 impairs cell migration of murine lung fibroblasts.

<p>(A) WT ACTN4 and ACTN4 KD murine lung fibroblasts grown in 6-well Petri-dish plates were lysed with RIPA buffer in the presence of 1× proteinase cocktail mix. Equal amount of total protein were separated by 7.5% SDS-PAGE and immunoblotted with anti-ACTN4, ACTN1 and pan-ACTN. The data are representative of three independent experiments. (B) Cells were plated on 6-well plastic dishes and grown to confluence in DMEM in the presence 10% fetal bovine serum. A scraped area was made by a rubber policeman and then cultured for additional 24 hr. Photographs were taken at 0 hr and 24 hr, and the relative distance migrated by the cells was determined by using Photoshop software. Data are mean ± s.e.m., measured from three independent experiments each in triplicate.</p

Knockdown of ACTN4 increases both contractile force and gel compaction generated by murine lung fibroblasts.

<p>(A) Contractile force assays were performed with both WT ACTN4 and ACTN4 KD murine lung fibroblasts transiently transfected with either eGFP or human wild type ACTN4-eGFP. For each cell type, at least 20 individual cells with GFP fluorescence were analyzed. Quantification represents the average traction force per cell (± the standard deviation) of at least 20 individual fluorescent cells chosen randomly. Shown are representative of at least independent experiments. (B) Images created by using special software. The length of arrows stands for the strength of traction force. Shown are representative images of three independent experiments. (C) Immunoblotting of MLC2 of total cellular lysate and actin as loading control. The bands of MLC2 were quantitated by using ImageJ software. Shown are representative blot of three independent experiments. Data are mean ±., measured from three independent experiments. (D) Immunofluorescence of MLC2 in both ACTN4 and ACTN4 KD murine lung fibroblasts. Images are representative of cells chosen randomly from three independent experiments. Quantification represents the average fluorescence (± the standard deviation) of at least 50 individual fluorescent cells chosen randomly. Bar  = 10 µm. (E) A compaction assay was performed with WT ACTN4 and ACTN4 KD fibroblasts. Data stands for the area of gel top surface after 24 hr incubation. Data are mean ±., measured from three independent experiments. (F) Immunoblottings of MYH9 and MYH10 of total cellular lysate and actin as loading control. Shown are representative blots of three independent experiments. (G) Quantitative PCR (see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013921#s2" target="_blank">methods and materials</a>” for details). Data are mean ±. s.e.m., measured from three independent experiments each in triplicate.</p

Knockdown of ACTN4 increases both cellular and nuclear cross-sectional area of murine lung fibroblasts.

<p>Murine lung fibroblasts of WT ACTN4 and ACTN4 KD transiently expressed eGFP and ACTN4 KD transiently expressed ACTN4-eGFP or ACTN1-eGFP were trypsined and then plated in 6-well Petri-dish plates coated with 1 µg/ml fibronectin. After incubation for 16 hr, cells were further incubated with 10 µg/ml Hoechst 33342 for 10 min at 37°C in 5% CO₂ incubator prior to fixation with 2% formaldehyde for 30 min at room temperature. After taking fluorescent images under microscope, the cellular cross-sectional area of attached cells with GFP fluorescence (A) and the nuclear cross-sectional area with blue fluorescence (B) were measured by using ImageJ software. Quantification represents the average cellular and nuclear cross-sectional area (± the standard deviation) of at least 50 individual cells chosen randomly.</p

Knockdown of ACTN4 decreases cell proliferation.

<p>Both WT ACTN4 and ACTN4 KD fibroblasts were seeded at a density of 40K cells per well in 6-well Petri-dish plates. After culturing for appropriate time, all cells were harvested and only viable cells were counted on a hemacytometer. Data are the mean ± s.e.m., measured from three independent experiments each in triplicate.</p

Knockdown of ACTN4 hastens cell spreading of murine lung fibroblasts.

<p>(A) Murine lung fibroblasts (WT ACTN4 and ACTN4 KD) were transiently transfected to express the designated constructs. These cells were trypsined and then plated in 6-well plates coated with 1µg/ml fibronectin. After culturing for 5 min, 10 min, 15 min, 30 min, 1 hr, 2 hr and 16 hr, respectively, unattached cells were washed away with PBS. Adhesive cells were fixed with 2% formaldehyde for 30 min at room temperature followed by imaging under fluorescent microscope. The cellular cross-sectional area of attached cells with GFP fluorescence was measured by using ImageJ software. Quantification represents the average cellular cross-sectional area (± the standard deviation) of at least 50 individual fluorescent cells chosen randomly. (B) Immunoblotting of endogenous ACTN4 and exogenous human wild type ACTN4-eGFP. The immunoblotting results were the representative of three independent experiments. (C) Morphology of both WT ACTN4 and ACTN4 KD fibroblasts grown in Petri dishes and the quantification of protrusions per cell. Images present both WT and KD fibroblasts chosen randomly. Quantification represents the average protrusions per cell (± the standard deviation) of at least 50 individual cells chosen randomly. Bar  = 5 µm.</p

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

DNA pooling strategy for 960 individuals with low (n = 480) vs. high (n = 480) urinary Ca²⁺ excretion.

<p>NHS = Nurses' Health Study; HPFS = Health Professional Follow-up Study.</p

Distribution of all identified Claudin14 SNPs in the low and high urinary Ca²⁺ excretion groups with unadjusted Chi square P values.

<p>The missense variant rs113831133, highlighted in bold, was more frequent among individuals with lower urinary Ca²⁺ excretion. This association did not reach statistical significance when adjusted for multiple comparisons.</p

Candidate genes included in the resequencing study (n = 40).

<p>Candidate genes included in the resequencing study (n = 40).</p