Journal: PLoS ONE

Article Title: Deletion of suppressor of cytokine signaling 3 (SOCS3) in muscle stem cells does not alter muscle regeneration in mice after injury

doi: 10.1371/journal.pone.0212880

Figure Lengend Snippet: Tamoxifen-treated control (SOCS3 fl/fl Pax7-CreER - ) and SOCS3 MKO (SOCS3 fl/fl Pax7-CreER + ) mice were left uninjured (UN) or received a 40 μL injection of notexin (10 μg/ml) into the right TA muscle. Mice were killed for analysis at 1 (D1), 2 (D2), 3 (D3), 7 (D7) or 14 days (D14) post-notexin injury. RNA was extracted from snap frozen muscles following dissection and qRT-PCR performed using primers to detect Pax7 (A), MyoD (B), and Myogenin (C). Protein was extracted from remaining OCT embedded right TA muscles after sectioning and western immunoblotting performed. Representative immunoblots for Pax7 (D), MyoD (E), Myogenin (F) and embryonic myosin heavy chain (G) protein levels are shown. Band intensity was quantified using ImageQuant software (Bio-Rad Laboratories) and normalized to total protein levels (TPS). Data are expressed as mean ± SEM. Statistical analysis was performed using a two-way ANOVA with a Bonferroni’s post-hoc multiple comparisons test to determine the effects of genotype and time. n = 5–6 mice/time-point/genotype. * P < 0.05, **** P < 0.0001 compared to control. (H) Representative Pax7 (left) and DAPI (right) immunostained sections of TA muscle from control and SOCS3 MscKO mice at D14 post-injury. The number of Pax7 positive nuclei (as indicated by arrows) was counted per field of view. (I) Representative black and white laminin and DAPI immunostained sections of TA muscle from control and SOCS3 MscKO mice at D14 post-injury. Muscle fibers containing 1, 2, or 3+ -located nuclei were counted per field of view. Data are expressed as mean ± SEM. Statistical analysis was performed using an unpaired Student’s t-test. Scale bar = 100 μm. n = 5–6 mice/time-point/genotype. * P < 0.05, *** P < 0.001 compared to control.

Article Snippet: The following primary antibodies were used throughout the experiments in 5% BSA/TBS/0.1% Tween-20: Rabbit-anti-phosphorylated STAT3 (Y705) (#9131; Cell Signaling Technology, 1:1000), Rabbit-anti-STAT3 (#4904; Cell Signaling Technology, 1:1000), Mouse-anti-myogenin (F5D; #sc12732; Santa Cruz Biotechnology Inc., Dallas, Texas, USA, 1:400), Mouse-anti-Pax7 (developed by A. Kawakami from the Tokyo Institute of Technology and obtained from the Developmental studies hybridoma bank, 1:100), Rabbit-anti-MyoD (M318; #sc760; Santa Cruz, 1:250), and Mouse-anti-MyHC embryonic (F1.652, developed by H. Blau from the Baxter Lab for Stem Cell Biology, Stanford University and obtained from the Developmental studies hybridoma bank, 1:1000).

Techniques: Injection, Dissection, Quantitative RT-PCR, Western Blot, Software