Characterization of ILK-deficient mutant cells expressing inactive αIIbα6Bβ3.

<p>(A) Immunoblotting for ILK, PINCH, α-parvin, talin, and kindlin-2. Cell lysates obtained from parental cells with constitutively active αIIbα6Bβ3, ILK-deficient mutant cells with inactive αIIbα6Bβ3, and mutant cells transiently transfected with rat ILK cDNA were electrophoresed on SDS-PAGE gels and immunoblotted with indicated Abs. GAPDH shows an internal loading control. (B) Flow cytometry analysis showing PAC-1 (an activation-specific mAb for αIIbβ3) binding to mutant cells transiently transfected with either ILK plasmid or empty plasmid. Bound PAC-1 was detected with a PE-conjugated secondary mAb. </p

Knockdown effects of PINCH, parvins, and kindlin-2 in αIIbα6Bβ3-active parental cells.

<p>αIIbα6Bβ3-active parental cells were transiently transfected with PINCH siRNAs (p157 and p755) (A), α-parvin siRNAs (pa503 and pa761) (C), β-parvin siRNAs (pb900 and pb1011) (C), kindlin-2 siRNAs (k770 and k1733) (E), negative control siRNAs, and scrambled siRNAs. Cell lysates were electrophoresed on SDS-PAGE gels, and the separated proteins were immunoblotted with the indicated Abs. GAPDH and β-actin are shown as internal loading controls. The activation indexes of transfected cells (B, D, F) were calculated using the formula shown in Materials and Methods. A value of 100% implies the maximum PAC-1 binding to the cells treated with dithiothreitol (DTT). Data represent means ± standard deviation (SD) of three (B, F) or four (D) independent experiments. ** indicates <i>P</i> < 0.01.</p

Detection of IPP complex proteins in αIIbα6Bβ3-active parental cells.

<p>Cell lysates obtained from αIIbα6Bβ3-active parental cells were immunoprecipitated with Abs against PINCH (A), α-parvin (B, C), and ILK (D). The co-precipitates were detected by Abs for α-parvin (A), ILK (B), and PINCH (C, D). IgG means immunoprecipitation (IP) using non-immune control IgG. IB stands for immunoblotting. Arrows indicate the predicted sizes of the indicated proteins. Arrowheads (D) indicate the antibody heavy chains used in the IP. Different mobilities between those of the two IgG antibodies are probably caused by differences in the amino acid compositions of them. </p

Effects of ILK mutants with defects in either PINCH or parvin binding.

<p>The activation indexes of transfected cells (A, C, E). ILK-deficient mutant cells were transiently transfected with GFP cDNA, GFP-fused wild-type ILK (GFPILK-WT) cDNA, GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) cDNA (A, E), or GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) cDNA (C, E). After transfection, the binding of either PAC-1 (A, C) or fibrinogen (E) to the cells was analyzed by flow cytometry. The activation index was determined by the formula shown in Materials and Methods. A value of 100% represents the maximal binding of PAC-1 or fibrinogen to the cells treated with dithiothreitol. Data represent means ± SD of three independent experiments. ** indicates <i>P</i> < 0.01. Immunoblotting showing protein expression of GFP (B, D), GFP-fused wild-type ILK (GFPILK-WT) (B, D), GFP-fused ILK mutant with defective PINCH binding (GFPILK-H99D/F109A/W110A) (B), and GFP-fused ILK mutant with defective parvin binding (GFPILK-M402A/K403A) (D) in ILK-deficient mutant cells. Cell lysates were electrophoresed and immunoblotted with indicated Abs. </p

Comparison of genetic or acquired abnormalities among Western countries and Japan.

<p>The frequency of each genetic or acquired abnormality in our aHUS cohort in Japan was compared to that in three other cohorts (Italy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref032" target="_blank">32</a>], USA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref033" target="_blank">33</a>], France [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124655#pone.0124655.ref034" target="_blank">34</a>]). The data reported by Noris et al. included 47 secondary aHUS patients. Sixty-percent of aHUS patients in our cohort were enrolled from West Japan. In the data of Japan, both predisposing and potentially predisposing mutations were counted. Although two patients (2V1 and H2) had two predisposing mutations (C3-p.I1157T and THBD-p.D486Y), they were counted as C3 group.</p><p>CFH: complement factor H, Ab: antibody, C3: complement component C3, MCP: membrane cofactor protein, THBD: thrombomodulin, CFB: complement factor B, CFI: complement factor I, ND: no data</p><p>Comparison of genetic or acquired abnormalities among Western countries and Japan.</p

Family 2A with a C3-p.I1157T mutation.

<p>(A) Patient 2A1 (male) had six bouts of atypical hemolytic uremic syndrome (aHUS), at the age of 9, 15, 18, 22 and 29 (twice). Gray squares or circle indicate individuals who were not analyzed in this study. (B) The hemolytic assay showed that neither patient (P) nor his parents (father: F and mother: M) had appreciable hemolysis. However, Patient 2A1 and his father carried the same predisposing mutation p.I1157T in C3, confirmed by restriction fragment length polymorphism (RFLP) analysis (C) and direct DNA sequencing.</p

Family 2K with no predisposing or potentially predisposing mutations.

<p>(A) Patient 2K1 (female) developed atypical hemolytic uremic syndrome (aHUS) at the age of 35. Her husband (gray square) was not analyzed in this study. (B) The hemolytic assay showed that plasma from the patient (P), her father (F), and two daughters (D1 and D2) induced severe hemolysis, which was not observed in her mother (M). (C) The enhanced hemolysis detected in these four family members was corrected by the addition of purified complement factor H (CFH). However, no predisposing or potentially predisposing mutations were detected in Patient 2K1.</p

Relationship between hemolytic activity and complement abnormalities in 45 patients with atypical hemolytic uremic syndrome (aHUS).

<p>The results of the hemolytic assay for 45 aHUS patients are shown according to the predisposing (bold type) or potentially predisposing mutations and/or the acquired abnormalities. The patients carrying two mutations were classified according to the predisposing mutation, and the other mutations were described alongside the predisposing mutations or black bars. Two patients (2V1 and H2) were categorized into the C3 group, although they had two predisposing mutations (C3-p.I1157T and THBD-p.D486Y). In two patients (2P1 and 3G1), the hemolysis induced by serum was represented by gray bars. The normal range of hemolysis (5.4±1.8%: mean ± standard deviation) obtained from 20 healthy individuals (10 males and 10 females) is shown at the bottom of Fig 3. The results of the restriction fragment length polymorphism (RFLP) analysis of C3-p.I1157T are summarized to the right of the hemolytic assay results. Regarding the RFLP analysis of C3-p.I1157T (c.3470T>C), the heterozygous mutation (T/C) showed two bands (278 bp, 314 bp) and the homozygous mutation (C/C) showed one band (314 bp). † H2 had a homozygous mutation in C3-p.I1157T.</p

Family 2I with a CFH-p.R1215Q mutation.

<p>(A) Patient 2I1 (male) had episodes of atypical hemolytic uremic syndrome (aHUS) at the age of 28 and 29. Gray squares or circles indicate individuals who were not analyzed in this study. (B) The hemolytic assay showed that plasma from the patient (P), his mother (M), and one brother (B1) induced severe hemolysis, whereas samples from his father (F) and the other brother (B2) showed no appreciable hemolysis. (C) The hemolysis detected in the three family members was corrected by the addition of purified complement factor H (CFH). Genetic analysis showed that the patient, his mother, and brother 1 carried a CFH-p.R1215Q mutation, but his mother and brother 1 have never had the episodes of aHUS.</p