Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

<p>Correction: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress</p

<i>Wwox</i> KO mice have abnormal spleens and thymuses.

<p>Histopathology of spleens (top four panels) and thymuses (bottom four panels) from 18 day old KO and WT mice. Histological sections were stained with H&E. Note the significant atrophy of the spleen and the reduced cortical thickness in the thymus from the KO mice. Low power images of spleens (first row) and thymuses (third row) have a total magnification of 21X. High power images of spleens in (second row) have a total magnification of 84X and thymuses (bottom row) have a total magnification of 44X.</p

Genotypes of pups from <i>Wwox^+/ΔCre</i> × <i>Wwox^+/ΔCre</i> crosses.

<p>Genotypes of pups from <i>Wwox^+/ΔCre</i> × <i>Wwox^+/ΔCre</i> crosses.</p

<i>Wwox</i> is strongly expressed in the kidney tubules.

<p>Kidneys from a WT (left panel) or a KO (right panel) 18 day old mouse were dissected, paraffin embedded and subjected to immunohistochemical staining using anti-WWOX antibody. The strongest staining structures represent the distal convoluted tubule section of nephrons. Photomicrographs were taken using a 40X objective.</p

Histomorphometric analysis.

<p><b>(A)</b> MicroCT images of femurs from postnatal day 18 mice. Note the dramatic difference in bone structure of the femur from KO mice vs WT and HET mice bones <b>(B)</b> Von Kossa stained sections of femurs from mice of the indicated genotypes. Black stained areas represent mineralized bone tissue. Note the significantly decreased mineralization in the KO mice bone sample <b>(C)</b> Quantitative histomorphometric analyses of postnatal day 18 day mice. We did not observe differences between wild-type and heterozygous mice, therefore, we compared KO data (n = 3) with WT and HET data combined (WT+HET, n = 5). Wild-type-WT, heterozygote-HET, knockout-KO. BV/TV%-bone volume/tissue volume, Md.V/TV%-mineralized volume/tissue volume, Tb.N-Trabecular number (1/mm), Tb.Th-Trabecular thickness (micrometers). *p<0.05.</p

Targeting the Mouse <i>Wwox</i> allele.

<p><b>(A)</b> Insertion of the targeting vector into the <i>Wwox</i> allele introduced a new Bgl II (B) restriction site into <i>Wwox</i> Intron 1. The new Bgl II site leads to a smaller Bgl II restriction fragment when analyzed by Southern blot with the external hybridization probe. Additionally, a Not I restriction site was engineered at the 3′ end of the right arm used to linearize the targeting construct prior to electroporation. <b>(B)</b> Southern blot genotyping of wild-type (wt/wt) mice, heterozygous mice (wt/flox) and homozygous mice (flox/flox). <b>(C)</b> Multiplex-PCR based genotyping using allele specific primers. Primers A and B anneal to <i>Wwox</i> specific sequences in the <i>Wwox</i> upstream regulatory region (primer A) and in <i>Wwox</i> Exon 1 (primer B). Primer C anneals to sequences in the LoxP <i>Cre</i>-recombination in the targeted <i>Wwox</i> allele. Multiplex PCR using primers A, B and C yield DNA products having sizes specific for the wild-type and targeted alleles.</p

<i>Wwox</i> KO mice have reduced postnatal growth.

<p><b>(A)</b> Photograph of WT and KO newborn littermates. <b>(B)</b> F2 pups were weighed on postnatal days 3, 10, 14 and 17. 100% of <i>Wwox</i> KO mice died before weaning (21 days). WT, n = 18; HET, n = 43; KO, n = 8. Error bars represent ±SEM.</p

Blood Chemistry Analysis.

<p>*Values are presented as the average±SEM.</p><p>**p-value using student's t-test comparing WT+HET vs. KO; NS-p>0.05.</p

BAG3 inhibits egress of infectious recombinant virus VSV-M40.

<p>HEK293T cells were first transfected with vector alone, BAG3-WT or BAG3-ΔN for 24 hours, and then infected with recombinant virus VSV-M40 <b>(A)</b> or VSV-M40-P2728A <b>(C)</b> at a MOI of 0.1 for 8 hours. Supernatants were harvested and virus titers were determined by standard plaque assay on BHK-21 cells. Each bar represents the average of three independent experiments performed in duplicate. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. The indicated proteins from VSV-M40 <b>(B)</b> or VSV-M40(P2728A) <b>(D)</b> infected cell extracts were detected by Western blotting.</p

Strategy for <i>Cre</i>-mediated ablation of <i>Wwox</i> expression.

<p><b>(A)</b> To promote deletion of <i>Wwox^flox</i> alleles we used transgenic mice carrying the <i>Cre</i>-recombinase gene under control of the adenoviral <i>EIIA</i>-promoter. <i>EIIA</i>-regulated <i>Cre</i>-recombinase is expressed in pre-implantation embryos leading to site-specific deletion of LoxP flanked (floxed) sequences in all tissues including germ cells. <b>(B)</b> PCR-based strategy to demonstrate <i>Cre</i>-recombinase deletion of the <i>Wwox</i> target sequences. <b>(C)</b> Wwox protein expression is abolished in <i>Wwox</i> KO mice. Total protein extracts from the indicated tissues were analyzed by immunoblotting using Wwox specific antibodies. Anti-actin was used as a loading control. WT-wild-type, HET-heterozygous, KO-knockout.</p