Journal: Aging Cell

Article Title: The African killifish: A short‐lived vertebrate model to study the biology of sarcopenia and longevity

doi: 10.1111/acel.13862

Figure Lengend Snippet: Aged and late‐life cohorts display distinct cellular and physiological characteristics. (a) qRT‐PCR analysis of atrogin1 and murf1 expression in 6‐week early life, 22‐week early life, 37‐week aged, and 52‐week late‐life male killifish. Levels are relative to 6‐week‐old fish and error bars represent ± SEM. (b) Western blot images for LC3 and p62, and total protein direct blue stain, on protein lysates from 6‐week, 22‐week, 37‐week, and 52‐week‐old male killifish. To accurately reflect the variation observed in LC3 and p62 levels, three represented samples from each stage are displayed. Quantification of LC3II/LC3I ratio (c) and p62 levels normalized to total protein (d). Error bars represent± SD. (e–h) Representative images of Pax7, conjugated wheat germ agglutinin (WGA) and Dapi labelling on muscle from 6‐week, 22‐week, 37‐week, and 52‐week‐old male killifish. Heat map displaying fiber size distribution is also included. (i) Total number of Pax7+/Dapi+ muscle stem cells/per fiber at each time point. Error bars represent ± SD. (j) Number of Pax7+/Dapi+ muscle stem cells/per small fiber at each time point. Error bars represent ± SD. (k) Number of Pax7+/Dapi+ muscle stem cells/per medium fiber at each time point. Error bars represent ± SD. (l) Number of Pax7+/Dapi+ muscle stem cells/per large fiber at each time point. Error bars represent ± SD. (m i –m iv ) Representative maximum projection images of endplates, labelled with synaptic vesicle glycoprotein 2A (green) to detect presynaptic termini, and α‐bungarotoxin (red) to visualize acetylcholine receptors in the postsynaptic termini, in 6‐week, 22‐week, 37‐week, and 52‐week muscle. Arrowheads indicate examples of denervated endplates, which are positive for α‐bungarotoxin (red) but not synaptic vesicle glycoprotein 2A (green). Inset shows enlarged views of predominant endplates presented at each time point with 6‐week, 22‐week, and 37‐week displaying innervated endplates, and 52‐week samples displaying denervated endplates. Scale bar for inset is boxed. ( n ) Percentage of denervated endplates in 6‐week, 22‐week, 37‐week, and 52‐week‐old male killifish. Error bars represent ± SD. (o) qRT‐PCR analyses of acetylcholine receptors ‐α ( chrna1 ), −β ( chrnb1 ), −γ ( chrng ), and −δ( chrnd ) in 6‐week, 22‐week, 37‐week, and 52‐week‐old male killifish. (p) qRT‐PCR analysis of myogenin ( myog ), myogenic factor 5 ( myf5 ), and myoblast differentiation protein 1 ( myoD ) in 6‐week, 22‐week, 37‐week, and 52‐week‐old male killifish. Levels are relative to 6‐week‐old fish and error bars represent ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001 calculated using one/two‐way ANOVA with Tukey's multiple correction post hoc test or a chi‐squared test.

Article Snippet: Primary antibodies used in this study include: anti‐collagen 1 (Abcam, ab23730, 1/300), anti‐pax7 (Santa Cruz, sc‐81,648, 1:100), anti‐synaptic vesicle glycoprotein 2A (SV2, DSHB, 1/10), anti‐myosin (labelling slow and fast Myosin, A4.1025, DSHB, 1/10), and anti‐slow myosin (F59, DSHB, 1/10).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Staining