Additional file 2: of Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Avidity index of pooled serums from Group 1 and Group 2, and neutralizing capacity of serums against hemolytic activity of venom of L. laeta. (A) Comparison of avidity index of pooled serums of Group 1 and Group 2 (1:100 diluted), treated with 6 M urea by IgG avidity ELISA. (ns) indicates not statistically significant. (B) Human erythrocytes were sensitized for 1 h at 37 °C with 25 μg/mL of venom of L. laeta in the presence or absence of pooled sera of Group 1 or Group 2 at 1:1, 1:10 and 1:100 dilutions, and evaluated in a complement-dependent hemolysis assay. Negative control was incubated only with VBS and with not presence of complement serum (control without complement). Results were expressed as percentage of hemolysis. The assays were made in duplicate for a total of two independent experiments and results are expressed as mean ± SEM. Significance was evaluated with an ANOVA one-way with Bonferroni post-hoc test; (ns) indicates not statistically significant. (TIF 9838 kb

Additional file 1: of Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Densitometry analysis for dot blot and Western blot shown in Figs. 1b, 2a and b. Intensity of dots and bands were realized using ImageJ program, verifying for non-saturation and subtracting background. (A) Values from dots of Fig. 1b were expressed as relative density percentage calculated for each dot and normalized against control dot intensity with anti-L. laeta venom antibodies. Values are means ± S.E.M (n = 3). In addition, values of Western blot from Fig. 2a and Fig. 2b were expressed as relative density calculated from area mean density of each band and (B) normalized against the control band with mouse anti-L. laeta venom serum, (C) or normalized against control band with pool of serums of Group 1. Significance was evaluated with an ANOVA one-way with Bonferroni post-hoc test; (ns) indicates not statistically significant, and *** indicates significant differences between dots and control with p < 0.05. (TIF 27016 kb

Additional file 3: of Heterophilic antibodies in sera from individuals without loxoscelism cross-react with phospholipase D from the venom of Loxosceles and Sicarius spiders

Detection of L. laeta venom by immunoblot using single serums of Group 1 and Group 2. (A – Right) SDS-PAGE in 12% gel of L. laeta venom stained with Coomassie brilliant blue. (A – Left) Immunoblot detection of L. laeta venom incubated with mouse L. laeta antivenom immune serum (1:10,000 dilution) (CP). Immunoblot incubated with pre-immune mouse serum (1:1000 dilution) (CN). (B) L. laeta venom immunoblot detected by single serum from loxoscelism group (Group 1). (C) L. laeta venom immunoblot detected by individual serum from without loxoscelism group (Group 2). (TIF 2986 kb

Homeotic Transformation Observed in the 4th Caudal Vertebra of <i>Hotair</i>^{<i>−⁄−</i>} mice.

<p>(A) Visualization of the sacral and caudal region of the mouse skeleton by microCT reveals a homeotic transformation in <i>Hotair</i>^{<i>−⁄−</i>} mice of the 4th caudal vertebra to a structure similar to that of the 3rd caudal vertebra. (B) Dorsal, lateral and ventral comparison of WT and <i>Hotair</i>^{<i>−⁄−</i>} 4th caudal vertebra reveals a structural abnormality in homozygotes indicative of a homeotic transformation.</p

Increased Expression of <i>Lincpint</i> from Postnatal Day 3 to 8 Weeks of Age.

<p><i>LacZ</i> reporter expression (blue) at 3 days, 3 weeks, and 8 weeks in F0 heterozygotes shows increasing <i>Lincpint</i> expression with age. (A) At 3 days, ßgalactosidase staining is only observed in portions of the brain, tendons and ligaments of the hind limb, and some bronchioles in the lung (arrow). (B) At 3 weeks, there is increased staining in the brain, hindlimb, atria of the heart, lung, and liver. (C) By 8 weeks of age, the whole brain, skeletal muscle of the hindlimb and chest, atria and myocardium, lung, and liver tissue all exhibit strong ß-galactosidase staining representative of increased <i>Lincpint</i> expression. Examples shown are representative of n>4 mice per group. (D) RT-PCR analysis of <i>GFP</i> in GA (gastrocnemius) muscle and kidney isolated from 8 week-old and 52 week-old wild type (WT), heterozygous (<i>Lincpint-GFP</i>^{<i>+⁄−</i>}) and homozygous (<i>Lincpint-GFP</i>^{<i>−⁄−</i>}) mice. (E) Comparison of endogenous <i>Lincpint</i> RNA level in GA (gastrocnemius) muscle and kidney of 8 week-old versus 52 week-old wild type (WT) tissues demonstrating increased in <i>Lincpint</i> gene expression. Examples shown are representative of n>4 mice per group.</p

<i>LacZ</i> Reporter Expression in Brains of 6–8 Week Old lincRNA Gene-Targeted F0 Generation Heterozygotes.

<p>The brain <i>lacZ</i> expression pattern (blue) for each lincRNA gene is as follows: (A<b>)</b><i>Crnde</i>, the colliculi (dorsal view, arrow); (B) <i>Pantr1</i>, neocortex, olfactory bulb, basal forebrain, and hypothalamus; (C<b>)</b><i>Pantr2</i>, neocortex, olfactory bulb, cerebellum, hypothalamus, and basal forebrain; (D<b>)</b><i>Lincenc1</i>, neocortex, parts of the cerebellum and medial hypothalamus, especially strong patterning in the olfactory projection and olfactory projection areas of the temporal cortex (ventral view, red arrow); (E<b>)</b><i>Celrr</i>, broadly in gray matter with the exception of the lateral cerebellum and ventral pons; (F) <i>Kantr</i>, possibly in deep cerebellar layers (dorsal view, star); (G<b>)</b><i>Lincpint</i>, ubiquitously in gray matter, especially intense in the hypothalamus; (H<b>)</b><i>Lincppara</i>, ubiquitously in gray matter, especially dense in the hypothalamus; (I<b>)</b><i>Peril</i>, midline of the hypothalamus (ventral view, arrowhead); and (J) <i>Tug1</i>, spinal cord gray matter and light gray matter in most structures except for the neocortex. n = 2, genotype confirmed male mice per lincRNA knockout project.</p

Abnormal Lung Morphology in <i>Fendrr</i> Knockout Mice at E13.5.

<p>(A) <i>LacZ</i> reporter gene expression at E12.5 in <i>Fendrr</i>^{<i>−⁄−</i>} embryos in the frontonasal region (FN) of the face, the aorta gonad mesonephros (AGM) region, and the respiratory tract, including the lungs (L) and trachea (T). (B) Dissection of lungs at E13.5 revealed an abnormal, disorganized, globular phenotype in the lobes of <i>Fendrr</i>^{<i>−⁄−</i>} embryos compared with <i>Fendrr</i>^{<i>+⁄−</i>}.</p

Aging-associated Phenotypes in <i>Lincpint</i> Knockout Mice.

<p>(A) <i>Lincpint</i>^{<i>−⁄−</i>} and <i>Lincpint</i>^{<i>+⁄−</i>} male mice exhibit a significantly slower growth rate than their wild type (WT) littermates and begin to show significant weight loss near 6 months of age. Data are plotted as the mean +/− SEM, n > 9 mice for each group. Significance was assessed by a one-way ANOVA (*, <i>P</i> < 0.05; **, <i>P</i> < 0.005; ***, <i>P</i> < 0.001). (B) Kaplan-Meier analysis of homozygous with heterozygous and WT mice. <i>Lincpint</i>^{<i>−⁄−</i>} male mice exhibit a significant reduction in survival compare to <i>Lincpint</i>^{<i>+⁄−</i>} and wild type littermates. Data are plotted as percent survival over 1 year observation. (C) Ventral and dorsal skin sections in <i>Lincpint</i>^{<i>−⁄−</i>} mice compared with <i>Lincpint</i>^{<i>+⁄−</i>} and WT littermates. (D, E, F, and G) MicroCT evaluation of body composition at 12-, 26- and 52-weeks of age. (D, E) Male <i>Lincpint</i>^{<i>−⁄−</i>} and <i>Lincpint</i>^{<i>+⁄−</i>} mice exhibit a significant reduction in body fat as early as 26-week of age. Female <i>Lincpint</i>^{<i>−⁄−</i>} mice have reduced body fat at an older age noticeably at 52-week of age (***, <i>P</i> < 0.001, one-way ANOVA). (F, G) A significant reduction in femur bone mineral density (BMD) observed in both males and females <i>Lincpint</i>^{<i>−⁄−</i>} compared with their <i>Lincpint</i>^{<i>+⁄−</i>} and WT littermates (*, <i>P</i> < 0.05; ***, <i>P</i> < 0.001, one-way ANOVA). (H) MicroCT images depict pronounced lordokyphosis (curvature of the spinal column) seen in older male and female <i>Lincpint</i>^{<i>−⁄−</i>} mice compared with WT littermates. (I) Approximately 70% (6/9 males and 7/10 females) of <i>Lincpint</i>^{<i>−⁄−</i>} mice have lordokyphosis by 12 weeks of age, compared with 0–20% of <i>Lincpint</i>^{<i>+⁄−</i>} (1/12 males and 2/10 females) and WT (1/10 males and 0/11 females) littermates. By 26 weeks of age the proportion of <i>Lincpint</i>^{<i>−⁄−</i>} mice with lordokyphosis increased to nearly 90% (7/8 males and 8/9 females) and appeared in approximately 60% (8/12 males and 6/10 females) of <i>Lincpint</i>^{<i>+⁄−</i>} mice, compared with less than 20% (2/10 males and 2/11 females) of WT littermates. n ≥ 9 mice per group for all observations reported.</p

Abnormal Hindlimb Posture, Reduced Grip Strength, and Muscle Wasting in <i>Hottip</i>^{<i>−⁄−</i>} Mice.

<p>(A) <i>Hottip</i>^{<i>−⁄−</i>} mice demonstrated unusual hindlimb clasping posture when suspended by the tail. (B) Cage endurance testing revealed that <i>Hottip</i>^{<i>−⁄−</i>} mice have a reduced ability to remain suspended from an inverted wire cage top, n = 5 mice for each group. (C) The right and left TA (tibialis anterior), GA (gastrocnemius) and Quad (quadriceps) muscles from WT, <i>Hottip</i>^{<i>+⁄−</i>} and <i>Hottip</i>^{<i>−⁄−</i>} mice were weighed. Muscle weights are normalized to body weight and calculated to include both right/left muscle weights. Data are means +/−SEM, n = 6 mice for each group. A significant decrease in muscle weight was observed only in the GA of <i>Hottip</i>^{<i>−⁄−</i>} animal in both males and females (male data not shown). Asterisks indicate a significant difference in the <i>Hottip</i>^{<i>−⁄−</i>} GA muscle weights compared to all other control groups (P< 0.01). (D) Comparison of GA muscle fiber numbers in WT, <i>Hottip</i>^{<i>+⁄−</i>} and <i>Hottip</i>^{<i>−⁄−</i>}. A significant reduction of fiber count was observed in <i>Hottip</i>^{<i>−⁄−</i>}. Significance assessed by one-way ANOVA (P < .0001 (E) Comparison of mean cross-sectional area of muscle fibers. Cross sections taken from the GA muscle were stained with an antibody against laminin and measured. There is no noticeable size difference between <i>Hottip</i>^{<i>−⁄−</i>} and control skeletal muscles, n = 6 mice per group for all muscle analyses.</p

Spatial <i>lacZ</i> Reporter Gene Expression in Mid-gestation Stage LincRNA Gene-Targeted Mouse Embryos.

<p>Heterozygous E12.5 embryos fixed and stained for ß-galactosidase showed a broad range of expression (blue) of the introduced <i>lacZ</i> reporter gene in the developing brain and craniofacial region (<i>Pantr1</i>, <i>Pantr2</i>, <i>Celrr</i>, and <i>Peril</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125522#pone.0125522.s002" target="_blank">S1 Fig</a>), neural tube (<i>Pantr2</i>, <i>Halr1</i>, and <i>Lincppara</i>), dorsal aorta (<i>Celrr</i>), heart (<i>Celrr</i>, <i>Peril</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125522#pone.0125522.s002" target="_blank">S1 Fig</a>), lungs (<i>Fendrr</i>), limb buds (<i>Hottip</i>, <i>HoxA11os</i>, and <i>Mannr</i>), foregut (<i>Hottip</i>, <i>HoxA11os</i>, and <i>Fendrr</i>), posterior region and the tail (<i>Hotair</i>, <i>Hottip</i>, and <i>HoxA11os</i>). <i>Tug1</i> showed a widespread <i>lacZ</i> expression pattern, whereas expression of other reporter genes was restricted to the epidermis (<i>Eldr</i>), mammary buds (<i>Lincenc1</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125522#pone.0125522.s002" target="_blank">S1 Fig</a>), or the whisker placode (<i>Trp53cor1</i>, see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125522#pone.0125522.s002" target="_blank">S1 Fig</a>). Examples shown are representative of at least five genotype-confirmed embryos per lincRNA knockout project.</p