Defects in gonadal integrity.

<p>n: Numbers of examined animals.</p>#<p>: Animals were raised at 15°C until examination.</p><p>P values were relative to:</p>a<p>: N2;</p>b<p>: <i>Is[daf-16::gfp]</i>;</p>c<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i>;</p>d<p>: <i>Is[daf-16::gfp]</i>(L4440);</p>e<p>: <i>shc-1(ok198)</i>;</p>f<p>: <i>shc-1(ok198)</i>(L4440);</p>g<p>: <i>shc-1(ok198);akt-1(ok525)</i>^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#nt115" target="_blank">#</a>};</p>h<p>: <i>akt-1(ok525)</i>;</p>i<p>: <i>shc-1(ok198);akt-1(ok525)</i>;</p>j<p>: <i>shc-1(ok198);daf-18(e1375);akt-1(ok525)</i>;</p>k<p>: <i>shc-1(ok198);akt-1(ok525)</i>;</p>l<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i> at 15°C.</p

Active DAF-16 derogates integrity of the gonadal basement membrane.

<p>(A) Knock-down of <i>daf-16</i> by RNAi suppressed the early lethality of shc<i>-1(ok198);Is[daf-16::gfp]</i> animals. Animals died within the first five days of adulthood: <i>shc-1(ok198);Is[daf-16::gfp](L4440)</i> as control: 45.7%, <i>shc-1(ok198);Is[daf-16::gfp]+daf-16</i> RNAi: 1.4%. (B) <i>daf-16</i> mutation suppresses the defects in gonad integrity of <i>shc-1</i> mutant animals. (C) Transgenic expression of a constitutively nuclearly located <i>daf-16(4A)::gfp</i> results in disruption of the gonad in <i>shc-1(−)</i>, but not in wild type animals (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s007" target="_blank">Figure S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>). (D) SHC-1 does not affect the subcellular localization of DAF-16(4A)::GFP. In both wild type and <i>shc-1(ok198)</i> animals DAF-16(4A)::GFP is constitutively located in the nucleus. The mean lifespan and statistical analyses in this figure are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t001" target="_blank">Table 1</a>. <i>byEx</i> represents extrachromosomal transgenic alleles which are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>. Mean values and statistic analysis of defect in gonadal integrity are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t002" target="_blank">Table 2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>. (E) SHC-1 does not affect expression level of transgenic <i>daf-16(4A)::gfp</i>. Columns represent pooled normalised values of three independent experiments plus standard deviation (SD). Mann Whitney test. Normalized GFP intensity: <i>byEx797</i>: 1.00±2.5 (n = 31); <i>shc-1;byEx797</i>: 1.00±2.1 (n = 33), P = 0.9670; <i>byEx798</i>: 1.00±0.19 (n = 37); <i>shc-1;byEx798</i>:1.06±0.22 (n = 48), P = 0.1591.</p

Lifespan of mutant strains.

<p>P values relative to</p>a<p>: <i>shc-1(ok198)</i>;</p>b<p>: <i>shc-1(tm1729)</i>;</p>c<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i>+L4440;</p>d<p>: N2 without FUDR;</p>e<p>: <i>Is[daf-16::gfp]</i> without FUDR;</p>f<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i> without FUDR;</p>g<p>: <i>mek-1(ks54)</i> without FUDR;</p>h<p>: N2 with FUDR;</p>i<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i> from L2 at 25°C;</p>j<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i>+L4440 with FUDR;</p>k<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i> at 15°C.</p>*<p>Animals were shifted to 25°C from L2 larval stage.</p

Gonad basement membrane phenotype of animals with transgenic <i>daf-16::gfp</i> or <i>daf-16(4A)::gfp</i>.

<p>Transgenes were coinjected with <i>rol-6</i>(pRF4) at 20 ng/µl.</p>*<p>: The animals carry only the injection marker <i>rol-6</i>.</p><p>The P-values were relative to:</p>a<p>N2,</p>b<p><i>shc-1(ok198)</i>,</p>c<p><i>Ex[daf-16::gfp]</i>,</p>d<p><i>Ex[daf-16(4A)::gfp]</i>.</p><p>n.d: not determined.</p

Lifespan of <i>daf-16</i> transgenic animals.

<p>(A) <i>shc-1(ok198);Is[daf-16::gfp]</i> animals show reduced lifespan. (B) Treatment with FUDR extends the lifespan of both <i>shc-1(ok198);Is[daf-16::gfp]</i> and <i>Is[daf-16::gfp]</i> animals. (C) Knock-down of <i>glp-1</i> extends lifespan of <i>shc-1(ok198);Is[daf-16::gfp]</i> animals (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s001" target="_blank">Figure S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s002" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s003" target="_blank">S3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s012" target="_blank">Table S1</a>). (D) The lifespan extension by FUDR does not require the adapter protein KRI-1. (E) In the absence of FUDR extrachromosomal <i>daf-16::gfp</i> transgene does not extend lifespan of wild type animals. (F) In the presence of FUDR extrachromosomal <i>daf-16::gfp</i> transgene extends lifespan of wild type animals. In the lifespan assay with FUDR treatment, animals were transferred onto the agar plates containing 0.1 mg/ml FUDR and <i>E.coli</i> 24 hours after the L4 larval stage. These were raised on the FUDR containing plates for four days and then transferred onto new plates without FUDR. The mean lifespan and statistical analyses in this figure are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t001" target="_blank">Table 1</a>.</p

Active DAF-2, AKT-2 and SGK-1 antagonize AKT-1 mediated inhibition of DAF-16 to derogate gonad integrity.

<p>(A) DAF-16 activity affecting gonad integrity is inhibited by AKT-1. (B) Inactivation of IIR DAF-2 suppresses the defect in the gonadal basement membrane (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s008" target="_blank">Figure S8</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s013" target="_blank">Table S2</a>). (C) Inactivation of <i>daf-2</i> extends lifespan of <i>shc-1(ok198);Is[daf-16::gfp]</i> animals. (D) <i>daf-2</i> mutation suppresses gonad disruption in <i>akt-1;shc-1</i> animals (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s009" target="_blank">Figure S9</a>). (E) AKT-2 and SGK-1 antagonize AKT-1. (F) <i>shc-1(ok198);sgk-1(ok538);Is[daf-16::gfp]</i> L3 larvea have less germ cells than <i>shc-1(ok198);Is[daf-16::gfp]</i> L3 larvea. <i>shc-1;Is[daf-16::gfp]</i> animals: 49±13 (n = 29); <i>shc-1;sgk-1;Is[daf-16::gfp]</i> animals: 33±6 (n = 23) (P<0.0001). (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s010" target="_blank">Figure S10</a>). The mean lifespan and statistical analyses in this figure are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t001" target="_blank">Table 1</a>. Mean values and statistic analysis of gonad disruption are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t002" target="_blank">Table 2</a>.</p

JNK signaling antagonizes hypodermal DAF-16 to ensure gonadal integrity.

<p>(A) <i>daf-18</i> mutation enhances disruption of the gonad in <i>shc-1(ok198);akt-1(ok525)</i> but not in <i>akt-1(ok525)</i> one day adult animals. (B) Knock-down of <i>mek-1</i> and <i>kgb-1</i> enhances the defect in <i>Is[daf-16::gfp]</i> animals. The mean values and statistic analysis are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t002" target="_blank">Table 2</a>. (C) Expression of hypodermal <i>daf-16(4A)::gfp</i> leads to disruption of the gonadal basement membrane. <i>byEx</i> represents extrachromosomal transgene alleles which are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>. Mean values and statistic analysis of defect in gonadal integrity are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>.</p

Additional file 1: Text S1. of Treating C3 glomerulopathy with eculizumab

Summary of genetic testing of patient C3GN3. Text S2. Summary of genetic testing of patient C3GN4. Text S3. Summary of genetic testing of patient DDD2. Table S1. Details on published cases with patient characteristics, and treatment response. Table S2. Details on treatment and response of patient C3GN1. Table S3. Details on treatment and response of patient C3GN2. Table S4. Details on treatment and response of patient C3GN3. Table S5. Details on treatment and response of patient DDD1. Table S6. Details on treatment and response of patient C3GN4. Table S7. Details on treatment and response of patient DDD2. Table S8. Details on treatment and response of patient C3GN5. (DOCX 155 kb