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 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

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

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

Relative muscle cross-sectional area and fiber number (mean, S.D.) for the hindlimb muscles of POUND and WT mice.

<p>Fiber number is calculated by dividing muscle CSA by the average fiber cross-sectional area.</p

Aromatic amino acid stimulated effects on signaling pathways under normoxic vs hypoxic conditions.

<p>Reverse-phase protein array graphs of two sets of BMMSCs grown under normoxic conditions (21% O₂/5% CO₂) and under low O₂ tension (3% O₂/5% CO₂; physiologic hypoxia). When cells were 80% confluent they were treated with Phe, Tyr or Trp (100 µM) in serum-free media for 3 h. The cells were washed twice with PBS and lysis buffer was added to the plates. a-Phe, b-Tyr and c-Trp. Results are expressed as means ± SEM for three independent experiments. *<i>p</i>≤0.05 and # <i>p</i>≤0.01. Percent change of hypoxia over control for Phe was determined as percent change of BMMSCs treated with Phe under hypoxic conditions (3% O₂%) vs. BMMSCs treated with Phe under normoxic conditions (21% O₂) (control = 100%). Both sets of BMMSCs were treated with Phe and grown under different oxygen levels. Same for Tyr and Trp pannels. RPS6: Ribosomal protein s6 (S6_pS240_S244-R-V). CAV1: Caveolin 1 (Caveolin-1-R-V). NDRG1: N-myc downregulated gene (NDRG1_pT346-R-V). AKT1 AKT2 AKT3: protein kinase B (Akt_pT308-R-V). STAT3: Signal transducer and activator of transcription 3 (STAT3_pY705-R-V). CTNNB1: β-catenin (beta-Catenin-R-V). NOTCH1: Notch homolog 1 (Notch1-R-V). MAP2K1: Mitogen activated protein kinase (MEK1-R-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1_pT37_T46-R-V). MAPK8: c-Jun N-terminal kinases (JNK_pT183_pT185-R-V). LCK: Lymphocyte-specific protein tyrosine kinase (Lck-R-V). CCNE1:Cyclin E1 (Cyclin_E1-M-V). BCl2: B-cell lymphoma 2 (Bcl-2-M-V). PIK3R1: Phosphatidylinositol 3-kinase regulatory subunit alpha (PI3K-p85-R-V). PKC: Protein kinase C (PKC-pan_BetaII_pS660-R-V). C12ORF5: TP53-inducible glycolysis and apoptosis regulator (TIGAR-R-V). PEA15: Phosphoprotein Enriched in Astrocytes 15 (PEA15_pS116-R-V). TGM2: transglutaminase2, C polypeptide, protein-glutamine-gamma-glutamyltransferase(Transglutaminase-M-V).</p

Hypoxia itself modulates BMMSC signaling pathways.

<p>Reverse phase protein arrays were performed under either normoxic or hypoxic conditions. <b>Panel A</b>: RPPA graph of two sets of untreated BMMSCs grown under normoxic conditions or low O₂ tension. When cells were 80% confluent they were switched to serum-free media for 3 h. Then cells were washed twice with PBS and lysis buffer was added to the plates. Results are expressed as means ±SEM for three independent experiments. *<i>p</i>≤0.05 and # <i>p</i>≤0.01. Percent change of untreated BMMSCs in hypoxia was determined as percent change of untreated BMMSCs in normoxia (control = 100%). RPS6: Ribosomal protein s6 (S6_pS240_S244-R-V). CAV1: Caveolin 1 (Caveolin-1-R-V). STAT3: Signal transducer and activator of transcription 3 (STAT3_pY705-R-V). AKT1 AKT2 AKT3: protein kinase B (Akt_pT308-R-V). CTNNB1: β-catenin (beta-Catenin-R-V). NOTCH1: Notch homolog 1 (Notch1-R-V). YBX1: The Y-box-binding Protein (YB-1-R-V). NRAS: Neuroblastoma RAS viral (v-ras) oncogene (N-Ras-M-V). MAP2K1: Mitogen activated protein kinase (MEK1-R-V). EGFR: Epidermal growth factor receptor (EGFR_pY1173-R-V). G6PD: Glucose-6 phosphate dehydrogenase (G6PD-M-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1-R-V). CDKN1B: Cyclin dependent kinase inhibitor (p27_pT198-R-V). EIF4EBP1: Eukaryotic initiating factor 4 (4E-BP1_pS65-R-V). MAPK8: c-Jun N-terminal kinases (JNK_pT183_pT185-R-V). TRFC: Total rosette forming cells CD71 marker (TRFC-R-V). PRKAA1: 5'-AMP-activated protein kinase catalytic subunit alpha-1 (AMPK_pT172-R-V). <b>Panel B</b>: RPPA heatmap showing up-regulation of Akt (more than one antibody), CAV1, STAT3 and NOTCH1 in untreated BMMSCs in hypoxia vs. untreated BMMSCs in normoxia (control).</p