Fig. 3
α-Klotho expression in quiescent and activated MuSCs. a Klotho expression in isolated MuSCs versus whole muscle lysates as per RNAseq analysis in transcripts per million (TPM)27 (n = 4/group; ****p < 0.0001, one-tailed Student’s t test). b Representative structured illuminescent microscopy of α-Klotho in young and old MPCs. Scale: 5 µm. c Quantification of α-Klotho in young and old MPCs (*p < 0.05, one-tailed Student’s t test). d ELISA analysis of α-Klotho in culture media alone, as well as conditioned media from young and old MPCs (n = 3/group; **p < 0.01, ****p < 0.0001, one-way ANOVA with Tukey’s post-hoc test). e Immunofluorescent colocalization of MyoD, F-actin, and α-Klotho 3 dpi. Scale: 50 µm. f Heat-map representation of α-Klotho as well as markers of MuSC activation (MyoD1, Fos, Jun, Myf5) in quiescent and activated cells from RNASeq analysis of publicly archived data from a recent report27. g Immunofluorescence staining of α-Klotho and DAPI in sorted MuSCs and fibroadipogenic progenitors (FAPs) fixed immediately after isolation (Day 0) or after activation in culture (Day 3). Scale: 12.5 µm. h Quantification of α-Klotho expression in MuSCs and FAPs at Day 0 and Day 3 of culture (****p < 0.0001, two-way ANOVA with Tukey’s post-hoc test). i Quantification of α-Klotho in the conditioned media of MuSCs and FAPs sorted from uninjured muscle. Conditioned media was obtained after 3 days in culture (n = 3/group; ****p < 0.0001, one-way ANOVA with Tukey’s post-hoc test). j Quantification of α-Klotho in MuSCs and FAPs isolated from uninjured muscle and muscle 3 dpi (**p < 0.01, ***p < 0.001, two-way ANOVA with Tukey’s post-hoc test). k Immunofluorescence imaging of α-Klotho and DAPI in MuSCs and FAPs sorted from uninjured muscle and muscle 3 dpi. Scale: 50 µm. l Immunofluorescent imaging of Pax7 and MyoD in MPCs isolated from wild-type and Kl+/− mice. Scale: 50 µm. m Quantification of the % of MyoD+ cells in MPCs from wild-type and Kl+/− mice (*p < 0.05, one-tailed Student’s t test). n Quantification of the % of Pax7+ cells in MPCs from wild-type and Kl+/− mice (One-tailed Student’s t test). o Immunofluorescent imaging of MyoD and DAPI in the injured muscles of nontargeting control (NTC) and shRNA to α-Klotho 14 dpi. Scale: 25 µm. p Quantification of the percentage of MyoD+ nuclei within the injured muscles of nontargeting control (NTC) and shRNA to α-Klotho 14 dpi (n = 4–6/group; ****p < 0.0001, one-tailed Student’s t test). Data represented as mean ± SEM
