Supplementary Figure 1. Satellite cell contribution to myofibers in whole. gastrocnemius/plantaris/soleus, diaphragm, and EOM of 12 or 20 month

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1 Keefe et al. p. 1

2 Supplementary Figure 1. Satellite cell contribution to myofibers in whole muscles. (a-l) Representative cross-sections through whole TA/EDL, gastrocnemius/plantaris/soleus, diaphragm, and EOM of 12 or 20 month Pax7 CreERT2/+ ;Rosa mtmg/+ mice showing GFP+ myofibers to which satellite cells have contributed. (a, d, g, j) No tamoxifen controls at 20 months of age show few GFP+ myofibers (n = 2, for all muscles). In no tamoxifen controls at 20 months the average %GFP myofibers/total myofibers is 0, 0.1, 0.4, 1.1, 6.5, 5.3, 1.6 for EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM, respectively (b, e, h, k) sections showing GFP+ myofibers to which satellite cells have contributed 6 12 months (n = 4, for all muscles). Inset in (d) section through gastrocnemius of Rosa mtmg/+ mice without tamoxifen shows no GFP+ myofibers (n=1). (c, f, i, j) sections showing GFP+ myofibers to which satellite cells have contributed 6 20 months (n = 6, for all muscles). EDL and soleus outlined with white solid line. Plantaris outlined with white dotted line. (a-l) Scale bars = 500 μm. (d inset) Scale bar = 100um. Keefe et al. p. 2

3 Supplementary Figure 2. Satellite cell contribution varies amongst different animals. Graph of the %GFP myofibers, indicating myofibers receiving satellite cell contribution, in 5 limb muscles, diaphragm, and EOM of 6 individual Pax7 CreERT2/+ ;Rosa mtmg/+ mice that had received TAM at 6 months and were harvested at 20 months. Satellite cells in mouse contributed to significantly more myofibers in all muscles compared with animals 17932, 17933, (p < , with Bonferroni correction). Satellite cells in mouse contributed to significantly fewer myofibers in all muscles compared with animals 17758, 17776, 17778, (p < , with Bonferroni correction). Statistical methods described in Methods. Keefe et al. p. 3

4 Supplementary Figure 3. Average myofiber CSA area declines in most limb muscles with age. (a) Average myofiber CSA of all myofibers in EDL, TA, gastrocnemius, plantaris, soleus, and EOM as well as soleus fast myofibers (MyHCI-), soleus slow myofibers (MyHCI+) diaphragm, diaphragm fast myofibers (MHCI-), diaphragm slow myofibers (MHCI+) at 12 (dark blue, n= 7 for each muscle) and 20 months (light blue, n = 4 for EDL, n = 5 for other muscles). Data are expressed as the mean of fiber sizes for individual animals ± 1 s.e.m. P-values were calculated using a generalized linear regression model with random effects used to control for variance within individual animals. Significance (p<0.05) is denoted with a yellow background. Note that the average myofiber CSAs are based on MuscleQNT analyses using the 12 month parameters (as detailed in Supplementary Table 2). In the EDL, TA, gastrocnemius, plantaris, and diaphragm the value for the CSA of the 20 month control differs from those shown in the 20 month control in Supplementary Figure 5a because 12 month and 20 month parameters used for MuscleQNT analyses differ for these muscles. (b) Total muscle cross-sectional area Keefe et al. p. 4

5 of EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM at 12 (dark blue, n= 7 for each muscle) and 20 months (light blue, n = 4 for EDL, n = 5 for other muscles). Data are expressed as the mean of the total muscle areas for individual animals ± 1 s.e.m. P-values were calculated using a two-tailed student t-test. Significance (p<0.05) is denoted with a yellow background. Total muscle area for gastrocnemius is for medial and lateral heads only. Keefe et al. p. 5

6 Supplementary Figure 4. Satellite cells are efficiently ablated in Pax7creERT2/+;RosaDTA/+ mice. (a-g) Representative cross-sections showing Pax7+ satellite cells in EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM at 12 and 20 months in Pax7creERT2/+;Rosa DTA/+ mice (see Figure 1 for similar representative cross-sections through Pax7+/+;Rosa DTA/+ mice). Scale bar = 100 μm. Mean number of Pax7+ satellite cells per volume (mm3) of muscle for Pax7+/+;Rosa DTA/+ mice and Pax7creERT2/+;Rosa DTA/+ mice at 12 months (dark blue, Pax7+/+, n = 6 TA, EDL, n = 7 other muscles; red, Pax7creERT2/+, n = 8 EDL, TA, diaphragm, n = 7 other muscles) and 20 months (light blue, Pax7+/+, n = 3 EDL, n = 5 other muscles; pink, Pax7creERT2/+: n = 4 EDL, n = 6 other muscles). Control histograms are the same as those shown in Figure 1. Data are expressed as mean ± 1 s.e.m for all graphs. Two-tailed student t-test, ***p< Keefe et al. p. 6

7 Supplementary Figure 5. Testing role of satellite cells in maintaining adult, uninjured muscle. (a, c) Average myofiber size of EDL, TA, gastrocnemius, plantaris, soleus, soleus fast myofibers (MyHCI-), soleus slow myofibers (MyHCI+), diaphragm, diaphragm fast myofibers (MyHCI-), diaphragm slow myofibers (MyHCI+), and EOM at 12 months (a) or 20 months (c) with satellite cell ablation Pax7 creert2/+; Rosa DTA/+ (12 months n = 8 for each muscle; 20 months n = 6 for each muscle) or in control Pax7 +/+; Rosa DTA/+ mice (12 months n = 7 for each muscle; 20 months n = 4 for EDL, n = 5 for other muscles). Data are expressed as the mean of fiber sizes for individual animals ± 1 s.e.m. P-values were Keefe et al. p. 7

8 calculated using a generalized linear regression model with random effects used to control for variance within individual animals. Note that the average myofiber CSAs are based on MuscleQnNT analyses using the 12 month (a) or 20 month (c) parameters. The 20 month control TA, gastrocnemius, plantaris, and diaphragm myofiber CSAs shown in (c) differ slightly from those shown in Supplementary Figure 3a in which 12 month parameters were used (and the 12 month parameters differ from the 20 month parameters in these particular muscles; see Supplementary Table 2.). (b, d) Total muscle area of EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM at 12 months (b) compared to 20 (d) months of age. Data are expressed as the mean of the total muscle areas for individual animals ± 1 s.e.m. P-values were calculated using a two-tailed student t-test. Significance (p<0.05) is denoted with a yellow background. Total muscle area for gastrocnemius is for medial and lateral heads only. Keefe et al. p. 8

9 Supplementary Table 1: Antibodies used in study Antibody Type Source Product Number Working Concentration Antigen Retrieval Secondary and Amplification PAX7 Mouse IgG1 DSHB PAX7 2.4μg/ml yes biotin goat α mouse IgG1, ABC, TSA MyHCI Mouse IgG1 Sigma M8421 (NOQ7.5.4D) 1.5μg/ml yes biotin goat α mouse IgG1, ABC, TSA LAMININ Rabbit polyclonal Sigma L μg/ml yes biotin goat α rabbit, streptavidin Dylight488/594 or ABC, TSA GFP Chick polyclonal Aves Labs GFP μg/ml no Dylight488 goat α chick DsRed Rabbit Polyconal Clontech μg/ml No goat α rabbit Cy3 Keefe et al. p. 9

10 Supplementary Table 2: MuscleQNT parameters for each muscle at each age. Muscle Age (months) Erosion Box Size Off Set Min. Fiber Size (um2) Max. Fiber Size (um2) EOM EOM EDL EDL TA TA Gast Gast Plantaris Plantaris Soleus Soleus Diaphragm Diaphragm Keefe et al. p. 10