*2.8. Quantitative Real-Time PCR*

Total RNA was extracted from frozen muscle samples using a PureLink™ RNA Mini Kit (Invitrogen Canada, Burlington, ON, Canada). The quantification and purity of RNA was assessed using the A260/A280 absorption method. Total RNA (2 μg) was reverse transcribed using a Superscript II® Reverse Transcriptase Kit and random primers (Invitrogen, Burlington, ON, Canada). The reactions were incubated at 42 ◦C for 50 min and at 90 ◦C for 5 min. The real-time PCR detection of mRNA expression was performed using a Prism® (Graphpad, San Diego, CA, USA) 7000 Sequence Detection System (Applied Biosystems, Foster, CA, USA). The cycle threshold (CT) values were obtained for each target gene. The ΔCT values (normalized gene expression) were calculated as CT of target gene minus CT of the geometric means of three housekeeping genes (*Cyclophilin B*, β*-Actin* and *18S*). The relative mRNA level quantifications of target genes were determined using the threshold cycle (ΔΔCT) method, as compared to sham AAV-GFP. The primer sequences for all genes are found in Supplementary Table S2.

#### *2.9. Data Analysis and Statistics*

All statistical analyses were performed using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). Comparisons of initial body weight and body weight loss between sham-operated and CLP mice were performed using unpaired bilateral student *t*-tests (*p*-values < 0.05 were considered statistically significant). Comparisons of the effects of Parkin overexpression on parameters of interest were performed using two-way repeated measures analysis of variance (ANOVA) (except for comparisons of mitochondrial shape descriptors, as detailed below). Corrections for the multiple comparisons following two-way repeated measures ANOVA were performed with the two-stage step-up method of Benjamini and Krieger and Yekutieli (*q* < 0.1 was considered statistically significant). One-way ANOVA followed by the two-stage step-up method of Benjamini and Krieger and Yekutieli were used for the following comparisons: sham AAV-GFP vs. CLP AAV-GFP; sham AAV-GFP vs. CLP

AAV-Parkin; sham AAV-Parkin vs. CLP AAV-GFP; sham AAV-Parkin vs. CLP AAV-Parkin (except for comparisons of mitochondrial shape descriptors, as detailed below) (*q* < 0.1 was considered statistically significant). Differences for the median values of shape descriptors to assess mitochondrial morphology were assessed using a Kruskal–Wallis test followed by a Dunn's multiple comparisons test (adjusted *p*-values < 0.05 were considered statistically significant). The exact numbers of animals within each group in all figures are indicated in the figure legends.
