Ch a feature is also consistent with the wellestablished propensity of these b2-m isoforms to misfold and selfaggregate [15,16]. The ability of the three b2-m isoforms to form oligomeric structures in vivo was then explored by performing dot-blot analysis on lysates of worms using the A11 antibody that specifically recognizes the amyloid oligomers. The expression of wild type protein was accompanied by a small A11-positive signal, which became stronger in transgenic worms expressing the two variants (Figure 2D). The quantification of the A11-immunoreactivity indicated that the oligomerization significantly increased 4.8 and 4.3 fold in P32G and DN6 mutants, respectively, compared to WT (Figure 2E, p,0.01 vs. WT, one-way ANOVA). Immunofluorescence studies were carried out to visualize the b2-m in transgenic C. elegans strains. A b2-m-positive signal was observed in the vulva muscles and anal Emixustat (hydrochloride) biological activity sphincter muscle in the tail regions: it begun at larval stages of WT, P32G and DN6 animals (data not shown) and became maximal at day CAL 120 biological activity 1-adult age (Figure 3). No signal was detected in worms that were transfected either with the empty vector or alternatively in the head (data not shown). The constitutive expression of the wild type or variant b2m did not lead to the formation of amyloid fibrils, since no X-34 reactive deposits were detected in the vulva and tail muscles of 2 days-old transgenic worms (Figure S1). We also investigated whether the expression of the different isoforms of human b2-m resulted in specific toxic behavioural phenotypes. First of all, the effect on the larval growth was considered. Larval growth in C. elegans is known to be exponential;Figure 1. Genotype of C. elegans transgenic strains. (A) PCR genotyping of adult transgenic worms transfected with the empty vector (vector) or vectors for expression of wild type b2-m (WT), P32G or 7?9 truncated form (DN6). The expected size of PCR products (about 360 bp) was observed. (B) Human b2-m mRNA expression in different transgenic strains was normalized to worm cell division cycle 42 (cdc-42, GTP binding protein) as endogenous reference. Data are expressed as mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0052314.gC. elegans Models for b2-m AmyloidosisFigure 2. Human b2-m protein expression. (A) Representative dot blot of b2-m (polyclonal anti-human b2-m antibody) in transgenic worms and (B) quantification of b2-m immunoreactive bands. Data are mean values of density of immunoreactive bands/mg of protein 6 SE of three independent experiments (N = 6). (C) Representative western blot of b2-m in control worms (vector), wild type b2-m expressing worms (WT), and in nematodes expressing P32G (P32G) or DN6 b2-m isoform (DN6). Day 1 adult worms were collected, processed as described in Methods section, and equal amounts of proteins (40 mg) were loaded on each lane and immunoblotted with polyclonal anti-human b2-m antibody (Dako). (D) Representative dot blot developed by antibody recognizing oligomers (A11) in transgenic worms and (E) quantification of A11-immunoreactive bands. Data are expressed as mean of density of A11 immunoreactive bands/mg of protein 6 SE of three independent experiments (N = 9); *p,0.01 vs WT, according to one-way ANOVA. doi:10.1371/journal.pone.0052314.gtherefore the growth rate is constant within larval phases and, reached a plateau in late adulthood [28]. After synchronization, the numbers of worms were scored after 24, 48 and 72 hours that corr.Ch a feature is also consistent with the wellestablished propensity of these b2-m isoforms to misfold and selfaggregate [15,16]. The ability of the three b2-m isoforms to form oligomeric structures in vivo was then explored by performing dot-blot analysis on lysates of worms using the A11 antibody that specifically recognizes the amyloid oligomers. The expression of wild type protein was accompanied by a small A11-positive signal, which became stronger in transgenic worms expressing the two variants (Figure 2D). The quantification of the A11-immunoreactivity indicated that the oligomerization significantly increased 4.8 and 4.3 fold in P32G and DN6 mutants, respectively, compared to WT (Figure 2E, p,0.01 vs. WT, one-way ANOVA). Immunofluorescence studies were carried out to visualize the b2-m in transgenic C. elegans strains. A b2-m-positive signal was observed in the vulva muscles and anal sphincter muscle in the tail regions: it begun at larval stages of WT, P32G and DN6 animals (data not shown) and became maximal at day 1-adult age (Figure 3). No signal was detected in worms that were transfected either with the empty vector or alternatively in the head (data not shown). The constitutive expression of the wild type or variant b2m did not lead to the formation of amyloid fibrils, since no X-34 reactive deposits were detected in the vulva and tail muscles of 2 days-old transgenic worms (Figure S1). We also investigated whether the expression of the different isoforms of human b2-m resulted in specific toxic behavioural phenotypes. First of all, the effect on the larval growth was considered. Larval growth in C. elegans is known to be exponential;Figure 1. Genotype of C. elegans transgenic strains. (A) PCR genotyping of adult transgenic worms transfected with the empty vector (vector) or vectors for expression of wild type b2-m (WT), P32G or 7?9 truncated form (DN6). The expected size of PCR products (about 360 bp) was observed. (B) Human b2-m mRNA expression in different transgenic strains was normalized to worm cell division cycle 42 (cdc-42, GTP binding protein) as endogenous reference. Data are expressed as mean 6 SD of three independent experiments. doi:10.1371/journal.pone.0052314.gC. elegans Models for b2-m AmyloidosisFigure 2. Human b2-m protein expression. (A) Representative dot blot of b2-m (polyclonal anti-human b2-m antibody) in transgenic worms and (B) quantification of b2-m immunoreactive bands. Data are mean values of density of immunoreactive bands/mg of protein 6 SE of three independent experiments (N = 6). (C) Representative western blot of b2-m in control worms (vector), wild type b2-m expressing worms (WT), and in nematodes expressing P32G (P32G) or DN6 b2-m isoform (DN6). Day 1 adult worms were collected, processed as described in Methods section, and equal amounts of proteins (40 mg) were loaded on each lane and immunoblotted with polyclonal anti-human b2-m antibody (Dako). (D) Representative dot blot developed by antibody recognizing oligomers (A11) in transgenic worms and (E) quantification of A11-immunoreactive bands. Data are expressed as mean of density of A11 immunoreactive bands/mg of protein 6 SE of three independent experiments (N = 9); *p,0.01 vs WT, according to one-way ANOVA. doi:10.1371/journal.pone.0052314.gtherefore the growth rate is constant within larval phases and, reached a plateau in late adulthood [28]. After synchronization, the numbers of worms were scored after 24, 48 and 72 hours that corr.
