PMC

Continuous tamoxifen treatment prevents grow-back of residual Pax7-expressing satellite cells. (A) Quantification of Pax7-expressing nuclei in Pax7^fl/CreERT2 mice induced with tamoxifen before and after acute injury; n = 3, **P < 0.01. (B) Quantification of Pax7-expressing nuclei in Pax7^fl/CreERT2 mice with continuous tamoxifen application before and after acute injury; n = 3, **P < 0.01. (C) Pax7 expression is lost after tamoxifen-induced excision in Pax7^fl/CreERT2 mice (IP injection) but reoccurs after acute injury, Pax7 immunostaining is shown in green, and nuclei are counterstained with DAPI (in blue). Staining for α7-integrin is shown in red, and nuclei are counterstained with DAPI (in blue). (D) Pax7 deletion is only maintained when tamoxifen is continuously administered during regeneration of CTX-injured muscles, Pax7 immunostaining is shown in green, and nuclei are counterstained with DAPI (in blue). Staining for α7-integrin is shown in red, and nuclei are counterstained with DAPI (in blue). (E) Quantification of numbers of satellite cells (marked by expression of α7-integrin) in Pax7^fl/CreERT2 mice induced with tamoxifen before and after acute injury; n = 3. (F) Quantification of numbers of satellite cells (marked by expression of α7-integrin) in Pax7^fl/CreERT2 mice with continuous tamoxifen application before and after acute injury; n = 3, **P < 0.01. (Scale bar: 50 µm.)

Inactivation of Pax7 in adult satellite cells markedly impairs muscle regeneration. (A) Schematic showing the experimental regime for single injury used in this study. (B) H&E staining of tibialis anterior muscle 21 d after acute injury induced by CTX injection. Arrowheads point to fat depositions. (C) Immunostaining for perilipin (in green) demonstrates increased adipogenesis in Pax7^fl/CreERT2 mice, laminin staining is shown in red, and nuclei are counterstained with DAPI (in blue). (D) Schematic showing the experimental regime for double CTX injury used in this study. (E) H&E staining of tibialis anterior muscle 21 d after the second injury induced by CTX injection. Arrowheads point to fat depositions. (E’) A magnification of E. (F) Immunostaining for perilipin (in green) demonstrates increased adipogenesis in Pax7^fl/CreERT2 mice, laminin staining is shown in red, and nuclei are counterstained with DAPI (in blue). (Scale bar: 100 µm.)

Significantly impaired muscle regeneration with continuous tamoxifen treatment. (A) H&E staining 10 d after acute injury reveals markedly impaired regeneration following continuous tamoxifen treatment. (Scale bar: 100 μm.) (B) Immunostaining for laminin (in red) showing decreased numbers of fibers and also reduced fiber feret. Nuclei are counterstained with DAPI (in blue). (Scale bar: 100 μm.) (C) Quantification of the minimal fiber feret of muscles at 10 d after acute injury; n= 6, ***P < 0.001. (D) Quantification of the amount of regenerating fibers at 10 d after CTX injury; n = 6, *P < 0.01. (E) Immunostaining for smooth muscle actin (SMA, in red) demonstrating increased numbers of SMA-positive nuclei in Pax7^fl/CreERT2 animals compared with Pax7^fl/+ animals at 10 d after acute injury. Nuclei are counterstained with DAPI (in blue). (Scale bar: 100 μm.) (F) Quantification of SMA-positive nuclei relative to the total number of nuclei; n = 6, *P < 0.05. (G) Quantification of the numbers of interstitial cells, n = 4, ***P < 0.001.

Pax7 deletion results in cell-cycle arrest and precocious differentiation. (A) Infection of Pax7^fl/fl myoblasts with an adenovirus encoding the Cre gene leads to depletion of Pax7 expression and loss of Myf5 protein expression. (B) Infection of Pax7^fl/fl myoblasts with an adenovirus encoding the Cre gene leads to depletion of Pax7 expression. Pax7 immunostaining is depicted in green. Nuclei are counterstained with DAPI. (Scale bar: 100 µm.) (C) Ad-Cre-mediated Pax7 deletion results in growth arrest in primary myoblasts, relative to control (Ad-GFP) myoblasts; n = 3, **P < 0.01. (D) The siRNA-mediated depletion of Pax7 expression in satellite cells leads to reduced numbers of satellite cells (marked by M-Cadherin staining in green) after 72 h of culture. Nuclei are counterstained with DAPI. (Scale bar: 100 µm.) (E) Following depletion of Pax7 in satellite cells on single myofibers, the number of satellite cells per fiber is significantly reduced; n = 4, ***P < 0.001. (F) Numbers of clusters (three or more satellite cells on a fiber attached to each other) are reduced following treatment with Pax7 siRNA compared with scrambled control; n = 4, ***P < 0.01. (G) Increase in the number of single satellite cells on myofibers following 72 h in culture and treatment with Pax7siRNA; n = 4, ***P < 0.01. (H) Numbers of myogenin-positive satellite cells increase after 72 h in culture following depletion of Pax7 expression with Pax7 siRNA; n = 4, ***P < 0.01.