Figure 3.

Apln, Aplnr, and Tead1 expression dynamics in regenerating skeletal muscle

(A and B) Single-cell RNA-sequencing analysis of a notexin injury response in TA muscle adult mice. TA muscle samples from 0, 2, 5, and 7 days post-injury (d.p.i.) with n = 2-3 mice per time-point were analyzed from De Micheli et al. (2020) (A) UMAP projection of scRNAseq data demonstrating cell-type annotations of clusters using markers shown in Figure S3.

(B) Dot plots showing expression of Apln, Aplnr, and Tead1 by cell-type cluster and time-point. Dot size shows the frequency of cells expressing non-zero transcript level. Dot color shows average expression level.

(C and D) In vitro expression of Apln and Aplnr protein by immunofluorescence (C) and mRNA by qRT-PCR (D) in C166 endothelial cells (ECs) and C2C12 myotubes differentiated for 8days n = 5 for C166 ECs; n = 4 for C2C12 myotubes. Scale bar, 100 μm.

(E–I) Regeneration of TA muscles of adult WT mice after IM injection of glycerol analyzed at 0, 3, 7, and 14 days.p.i. by gene expression microarray and immunohistology.

(E) Experimental overview. (F and G) Apln and Aplnr mRNA levels from transcriptomic analyses, normalized and presented as fold-change relative to mean of 0 days.p.i. Data are mean ± SEM of n = 5 (0, 14 days.p.i.) and n = 6 (3, 7 days.p.i.) mice. (H) Representative images of CD31 and Laminin immunostaining in regenerating muscle samples at 0, 3, 7, and 14 days.p.i. Scale bar, 50 μm. (I) Quantification of CD31⁺ endothelial cells per cross-sectional area.

Data are mean ± SEM of n = 5 TA muscles. In (F–G) and (I), p values are reported by two-tailed unpaired t-test compared to 0 days.p.i.; with ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.