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

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

<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> 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

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

<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

MOESM1 of 14-3-3ζ loss leads to neonatal lethality by microRNA-126 downregulation-mediated developmental defects in lung vasculature

Additional file 1: Figure S1. Characterization of the ES cell line RRR334. A RT-PCR to confirm that the cell line traps 14-3-3ζ schematic view of the integration of the gene trap vector in the 14-3-3ζ gene as described in the legend of Fig. 1. The arrowheads indicate the primers for PCR. Endogenous 14-3-3ζ is expressed both in wild-type ES cell control, TC1, and the mutant cell line RRR334. The exogenous mutant allele exists only in the RRR334 cell line. B Determination of the integration site of the gene trap vector using PCR. Arrowheads indicate the primers for PCR. The numbers on each lane of the gel indicate the primer position in the 14-3-3ζ gene. “N” indicates negative control; “M” indicates marker. The 1636 and 506-bp marker sizes are shown. C Western blot analysis of 14-3-3ζ expression level in 8 week old female B6/129 mice mammary gland. Quantification of relative 14-3-3ζ expression level is shown below the 14-3-3ζ blot panel. Figure S2. Characterization of truncated 14-3-3ζ. A Western blot of lysate of MCF7 vector control transfectants (Vc) and two MCF7 transfectants of HA-tagged N-terminal fragment 139 amino acids of 14-3-3ζ [C-terminal deletion (ΔC1 and ΔC12)]. Cells were treated with DMSO or 50 nM MG132 for 4 h. Endogenous 14-3-3ζ was detected using 14-3-3ζ antibody, while the exogenous 14-3-3ζ C-terminal deletion fragment was detected using HA antibody. B 14-3-3ζ N-terminal fragment did not affect p-Mek1 and p-Akt levels. Western blot on lysates from the indicated transfectants were performed with indicated antibodies. β-Actin was used as loading control. C 14-3-3ζ N-terminal fragment did not affect proliferation in MCF-7 cells. MTT assay was performed on the three indicated transfectants. OD was measured at 570 nm and normalized to 650 nm. Figure S3. 14-3-3ζ expression in FVB/NJ and CD-1 14-3-3ζ+/+ and 14-3-3ζ−/− mice. A Analysis of 14-3-3ζ and β-actin in different organs in the CD-1 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ expression level is shown below the western panel. B Analysis of 14-3-3ζ and β-actin in different organs in the FVB/NJ 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ expression level is shown below the western panel. C Analysis of 14-3-3ζ, 14-3-3β, 14-3-3ε and β-actin in the liver, kidney and lungs from FVB/NJ 14-3-3ζ+/+ and 14-3-3ζ−/− mice by western blotting. Quantification of relative 14-3-3ζ, 14-3-3β, 14-3-3ε expression level is shown below the western panel

RAS-related GTPases <i>DIRAS1</i> and <i>DIRAS2</i> induce autophagic cancer cell death and are required for autophagy in murine ovarian cancer cells

<p>Among the 3 GTPases in the DIRAS family, <i>DIRAS3/ARHI</i> is the best characterized. <i>DIRAS3</i> is an imprinted tumor suppressor gene that encodes a 26-kDa GTPase that shares 60% homology to RAS and RAP. DIRAS3 is downregulated in many tumor types, including ovarian cancer, where re-expression inhibits cancer cell growth, reduces motility, promotes tumor dormancy and induces macroautophagy/autophagy. Previously, we demonstrated that DIRAS3 is required for autophagy in human cells. <i>Diras3</i> has been lost from the mouse genome during evolutionary re-arrangement, but murine cells can still undergo autophagy. We have tested whether DIRAS1 and DIRAS2, which are homologs found in both human and murine cells, could serve as surrogates to DIRAS3 in the murine genome affecting autophagy and cancer cell growth. Similar to DIRAS3, these 2 GTPases share 40–50% homology to RAS and RAP, but differ from DIRAS3 primarily in the lengths of their N-terminal extensions. We found that DIRAS1 and DIRAS2 are downregulated in ovarian cancer and are associated with decreased disease-free and overall survival. Re-expression of these genes suppressed growth of human and murine ovarian cancer cells by inducing autophagy-mediated cell death. Mechanistically, DIRAS1 and DIRAS2 induce and regulate autophagy by inhibition of the AKT1-MTOR and RAS-MAPK signaling pathways and modulating nuclear localization of the autophagy-related transcription factors FOXO3/FOXO3A and TFEB. Taken together, these data suggest that DIRAS1 and DIRAS2 likely serve as surrogates in the murine genome for DIRAS3, and may function as a backup system to fine-tune autophagy in humans.</p