Fig. 3: Transcriptional regulation and activation of the ISR in response to muscle specific loss of PolG exonuclease activity.

Unbiased assessment of RNA abundance was analysed from skeletal muscle of PolG^cont and PolG^mut short-term mice using bulk RNA-sequencing. Computational analysis of the data was performed including (A) hierarchical clustering of differentially expressed genes, (B) principal component analysis (PCA) between PolG^cont and PolG^mut and, (C) volcano plot depicting genes that were upregulated (red dots) and downregulated (blue dots) in PolG^mut muscle compared to PolG^cont—grey dots indicate non-significantly altered genes. (D) Specific identification of components driving global changes in principal component analysis. For each pathway, a one-sample t-test on each gene set GOBP was performed, with the adjusted P-values used to rank the pathways for each of the first 5 principal components. The pathways are labelled with FDR first, followed by the principal components (duplicated pathways are skipped) (red=increased, blue=decreased, white=no change). Enrichment analysis (KEGG) on genes demonstrates (E) pathways that were significantly upregulated between the genotypes and (F) pathways that were significantly downregulated between the genotypes. Analyses of transcriptomic data (A–F) were corrected using Benjamini-Hochberg FDR, n = 6/group. Fgf21 and Gdf15 mRNA expression as determined by qPCR in muscles from (G) short-term (PolG^cont n = 9, PolG^mut n = 10) and (H) long-term (PolG^cont n = 6, PolG^mut n = 4) PolG^cont and PolG^mut mice. Gene expression analysis was validated by ELISA for protein abundance of FGF21 and GDF15 in plasma in (I) short-term (PolG^cont n = 10, PolG^mut n = 9) and (J) long-term (PolG^cont n = 5, PolG^mut n = 5) PolG^cont and PolG^mut mice. Gene expression and ELISA data are presented as mean ± SEM, with P-value between control and mutant biological replicates determined by two-sided, unpaired two-tailed Mann-Whitney test (G–J). Source data for these figures are provided in the Source Data file.