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. 2022 Apr 2;14(7):1814. doi: 10.3390/cancers14071814

Figure 3.

Figure 3

Apelin protects myotubes from dexamethasone-induced atrophy. Four day-differentiated myotubes were exposed for 24 h to vehicle (Ctrl) or 10 μM dexamethasone (Dexa) and representative brightfield images are shown. Scale bar, 15 μm (A). In radioactive-based assays, we measured long-lived protein degradation of myotubes treated with vehicle or 10 μM Dexa for 36 h; it was induced by 25% by Dexa (B). Multiple t-tests, **** p ≤ 0.0001, n = 6. Dexa-treated myotubes have lower total protein content than controls (Ctrl) (C). Unpaired t-test, *** p ≤ 0.001, n = 6. Like in vivo, Q-PCR shows that MuRF1 expression is induced (D), while apelin expression is reduced (E) in myotubes treated for 24 h with 10 μM Dexa. TBP was used as housekeeping gene. Unpaired t-test, **** p ≤ 0.0001, n = 6. Four day-differentiated myotubes transfected with the empty vector pLKO, as control, or preproapelin-encoding plasmid for 24 h were treated with 10 μM Dexa for 24 h. Exogenous preproapelin partially restrained the Dexa-induced degradation of long-lived proteins (F). Ordinary one-way ANOVA, * p ≤ 0.05, n = 6, ** p ≤ 0.01. Myoblasts were transfected with a plasmid in which Firefly luciferase (FLuc) is under the control of a MuRF1 promoter, with another for Renilla luciferase (RLuc), under the control of thymidine kinase promoter to normalize the data (ratio 50:1) and with the empty vector pLKO, as control, or a preproapelin-expressing plasmid (Apln). After 24 h, myoblasts were treated with vehicle or with 10 μM Dexa for 24 h. Dexa-induced MuRF1 expression was reduced in myoblasts that express exogenous preproapelin (G). Ordinary one-way ANOVA, ** p ≤ 0.01, **** p ≤ 0.0001, n = 12. Unlike apelin 36, apelin 13 fully protects myotubes from dexamethasone-induced atrophy. We measured in blind the diameter of four day-differentiated myotubes exposed for 24 h to vehicle or 10 μM Dexamethasone (DEXA) in combination with vehicle or 10 nM apelin peptides of different lengths. Dexamethasone reduced myotube diameter as expected by about 25%. Only the presence of apelin 13 fully protected them, while apelin 36 alone even reduced myotube diameters (H). Kruskal–Wallis test, **** p ≤ 0.0001, n = 261–297.