A paired t-test was performed to compare the gene expression profiles of stromal cells in control and acute injury conditions.

This revealed 355 genes that exhibited significant alteration of expression where 219 were upregulated and 136 were downregulated. Genes were filtered with a significance cut-off of p (XLSX)</p

Stromal cells were enumerated based on relevant immunophenotypes (CD140a(+) Sca1(-), CD140a(-) Sca1(+) and CD140a(+)Sca1(+)) and BrDU positivity.

Typical flowcytometry panels from acute injury (day3), tenotomy (day 7) and denervation (day10) are shown in the upper panel (n = 10). BrdU positivity of each stromal subpopulation is calculated and presented in pie charts in lower panel. Diminutive amount of BrdU positivity was observed in the control samples and the highest proliferation in stromal cells was observed following acute injury. Stromal cells from the immobilization models exhibited limited BrdU positivity (n = 10 for each group). (TIF)</p

Stromal cells isolated from the control muscles and the 3rd day samples following acute injury.

Cells were sorted based on relevant immunophenotypes. Sorting efficiencies for each sub-population was assessed using flow cytometry. Lowest enrichment was observed in CD140a(+)Sca1(+) population in control samples (80%). (TIF)</p

Fig 2 -

Stromal cell subpopulations following acute injury (d 3), tenotomy (d 3 and d 7), and denervation (d 10) were selected for CD45, CD31, and CD11b negativity (gating strategy) and further analyzed for CD140a(+)/Sca1(−), CD140a(−)/Sca1(+), and CD140a(+)/Sca1(+) subpopulations compared to the control (A). Acute injury resulted in activation of the CD140a(−)/Sca1(+) and CD140a(+)/Sca1(+) subpopulations (B). Likewise, tenotomy resulted in activation of the CD140a(−)/Sca1(+) and CD140a(+)/Sca1(+) subpopulations on d 3 and d 7 (C), whereas denervation resulted in activation of the CD140a(+)/Sca1(−) and CD140a(+)/Sca1(+) subpopulations (D). Variations of mononuclear cell numbers in studied models and time points are presented (E). Relative representative abundance of the investigated subpopulations is presented as a pie chart (F). All experiments are conducted on freshly isolated cells (n = 5 for acute injury, n = 10 for immobilization models) and independently triplicated. Typical (median) flow cytometry panels are shown.</p

Unified list of the highest expressing (top 300) genes exhibiting the highest variation (FC>3) in response to injury in the three studied cell subpopulations.

This signature was annotated using the matrisome gene ontology classification presented in Fig 5. (XLSX)</p

S3 Fig -

Unsupervised distribution dendrogram of samples (all transcripts) are shown in A. Distribution of samples using significant genes (S1 Table) are presented in B. (TIF)</p

Fig 1 -

Hind limb mononuclear cells from time-course tenotomy and denervation muscles were isolated, enumerated, and normalized to the contralateral muscles for the indicated time points in rats and mice (circles for rats and triangles for mice, n = 5, A). Denervation induced less of an increase in the mononuclear cell population, lasting until week 3. Compared to controls, statistically significant increase in the mononuclear cell population was evident from week 1 to week 4 in tenotomy (+ and * symbols indicate statistical significance of p2, B). The representative images of the time-points selected for further evaluation in this study are presented in panel C. Fibrotic changes in tenotomy samples were evident on d 7. Sirius red staining showed marked deposition of collagens, along with atrophy. Denervation resulted in more severe atrophy without collagen deposition on d 10 (Sirius red staining in C). BrdU staining showed extensive positive nuclei in muscles subjected to acute injury on d 3, but limited positivity in both denervation and tenotomy immobilized muscles. The positive nuclei were exclusively limited to the stromal compartment outside the laminin demarcated area (arrowheads, red staining, C). BrdU incorporation into the stromal mononuclear cells are investigated via flow cytometry (D) and the time-course expression of Col1a1 and Col1a2 is documented to show collagen deposition in models (E).</p

Upregulated gene list was annotated using the DAVID tool that revealed two major annotation clusters with enrichment scores of 26.50 and 24.73, both containing genes of secreted extracellular matrix components.

Upregulated gene list was annotated using the DAVID tool that revealed two major annotation clusters with enrichment scores of 26.50 and 24.73, both containing genes of secreted extracellular matrix components.</p

Percentage abundance (average of triplicated experiments) of the investigated subpopulations.

Percentage abundance (average of triplicated experiments) of the investigated subpopulations.</p

S4 Fig -

Stromal cells were sorted based on CD140a(-) Sca1(+) immunophenotype and were cultured to confluence (upper panel). Upon reaching confluence, cells were subjected to adipogenic or myogenic differentiation and observed up to 10 days. No myofibers was observed along the observation period. Lipid accumulation could be observed in some of the cells (mid panel). On the contrary, induction of adipogenic differentiation could be robustly induced in the majority of the cells (lower panel). (TIF)</p