SDF-1β preserves BMSC nuclear morphology following exposure to H₂O₂.

<p>Representative fluorescence micrographs of Hoechst 33342-stained A) Tet-Off-SDF-1β BMSCs and B) Tet-Off-EV control BMSCs after vehicle control or H₂O₂ treatment. Overexpression of SDF-1β in Tet-Off-SDF-1β BMSCs allows for a greater number of surviving cells and cells with preserved nuclear morphology after H₂O₂ treatment compared to Dox-suppressed and Tet-Off-EV controls (6 h, ±100 ng/ml Dox, ±1.0 mM H₂O₂, 20×, 40×, bar 100 µm, n = 3, 3 independent experiments).</p

SDF-1β preserves BMSC morphology following exposure to H₂O₂.

<p>Representative phase contrast micrographs of A) Tet-Off-SDF-1β BMSCs and B) Tet-Off-EV control BMSCs after vehicle control or H₂O₂ treatment. Overexpression of SDF-1β in Tet-Off-SDF-1β BMSCs allows for a greater number of cells with preserved morphology after H₂O₂ treatment relative to Dox-suppressed and Tet-Off-EV controls (6 h, ±100 ng/ml Dox, ±1.0 mM H₂O₂, 20×, 40×, bar 100 µm, n = 3, 3 independent experiments).</p

SDF-1β increases the number of surviving BMSCs following exposure to H₂O₂.

<p>Cell number of trypan blue-stained A) Tet-Off-SDF-1β BMSCs and B) Tet-Off-EV control BMSCs after vehicle control or H₂O₂ treatment. SDF-1β significantly increased the number of surviving cells (trypan blue negative) and decreased the number of dying cells (trypan blue positive) in response to H₂O₂ treatment compared to Dox-suppressed and Tet-Off-EV controls (6 h, ±100 ng/ml Dox, ±1.0 mM H₂O₂, ***p<0.0001, −Dox; H₂O₂ vs. +Dox; H₂O₂, n = 3, 3 independent experiments).</p

SDF-1β does not affect BMSC proliferation.

<p>Colorimetric quantification of DMSO-solubilized MTT formazan at 540 nm showed no differences in proliferation of Tet-Off-SDF-1β compared to Dox-suppressed and Tet-Off-EV controls (1,3, and 7 d, ±100 ng/ml Dox, n = 6, 3 independent experiments).</p

Primary myoblasts from POUND mice show impaired proliferation and differentiation.

<p>A. Primary myoblasts cultures from wild-type and POUND mice show a significant decrease in the proliferation and metabolic activity of myoblasts in POUND mice compared to normal wild-type mice as measured using MTS assay (left panel). B. Myoblasts from POUND mice (right panel, top micrograph) fail to differentiate normally and after 7 days do not develop into the elongate myotubes characteristic of normal, wild-type mice (right panel, bottom micrograph). C. Real-time PCR data show that that the early marker of myoblast differentiation, MyoD (left graph), and the later differentiation marker myogenin (right graph) are both significantly downregulated in myoblasts from POUND mice.</p

Altered leptin signaling in POUND mice alters IGF-1 signaling in skeletal muscle.

<p>A. ELISA assays show that muscle-derived IGF-1 is significantly decreased in the hindlimb muscles (extensor digitorum longus) from leptin receptor-deficient POUND mice (left graph), whereas protein levels of myostatin in hindlimb muscle are significantly elevated in POUND mice (right graph). B. Integrated pathway analysis from mRNA array comparing gene expression in tibialis anterior muscles of POUND mice with that of normal mice. The vertical axis represents the probability that a particular gene is associated with a specific canonical pathway by chance, the higher the score on this axis the lower the probability the association between gene and pathway is by chance alone. The strongest association revealed by the analysis is between genes altered in POUND mice and those associated with IGF-1 signaling. The open blue boxes connected by the lines represent ratio values indicating the ratio of genes detected in the pathway to the total number of genes in that particular pathway. C. Heat map from reverse phase protein analysis comparing protein expression in hindlimb muscle of POUND mice with that of normal mice. Arrows indicate proteins including Akt, MAPK, and MEK that are highly expressed in muscle from normal mice (red) but not highly expressed in muscle from POUND mice (green). Western blots shown on the right are for total and phosphorylated Akt, MAPK, and MEK.</p

Leptin increases myoblast proliferation.

<p>A. Leptin-treatment (100 ng/ml) significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 12 months of age. B. Leptin-treatment (100 ng/ml) also significantly increases cell proliferation and metabolic activity measured using MTS assay in primary myoblasts from mice 24 months of age. C. -treatment (100 ng/ml) significantly increases the expression of the myogenic factors MyoD and myogenin in primary myoblasts from mice 24 months of age. Leptin did not increase the expression of these factors in myoblasts from mice 12 months of age. D. Box-and-whisker plots showing ΔΔCt values (y-axis) for leptin (LEP) and leptin receptor (LEPR) expression in the soleus (SOL; top row) and extensor digitorum longus (EDL; bottom row) muscles of mice 12 and 24 months of age (x-axis). The whiskers mark the minimum and maximum values, the boxes the first and third quartiles, and the bar within the box indicates the median. Expression of the leptin receptor is not increased with age, and is significantly (P<.05) downregulated in aged soleus (SOLLEPR).</p

ZetaView measurements of the exosomes extracted from three different serum starting volumes of six individual human donors samples using the 4 different isolation techniques.

<p>NTA was done using the ZetaView instrument to measure (A) particles diameters (nm) and (B) the log10 of the total number of particles isolated using miRCURY (blue), ExoQuick (green), TEIR (red), and UC (yellow) from 1 ml, 500 μl, and 100 μl of human serum. The data in this graph are the mean values of the six individual human samples (±SEM), *p≤0.05.</p

Characterization of exRNA extracted from exosomes isolated using the four techniques.

<p>exRNA quality was evaluated using the Agilent Bioanalyzer with RNA 6000 Pico kit for the exosomes extracted using the different isolation techniques and serum volumes. The y-axis represents fluorescence, and the x-axis is the size of the RNA, measured in nucleotides (nt). (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.</p

Correlation between the volume of serum and the zeta potential of the isolated solutions.

<p>The data in this graph are the mean values of three experimental replicates (n = 3) ± SEM. (A) miRCURY, (B) ExoQuick, (C) TEIR, and (D) UC.</p