Fig 2: ULK1 is O-GlcNAcylated in cardiomyocytes. (a) Left panel, western blot analysis of Ad-LacZ- and Ad-Cre-infected Ogtf/f and/or Ogtf/y neonatal cardiomyocytes, using antibodies against O-GlcNAc and ULK1. GAPDH was detected as a loading control. Right panel, quantification of western blot results in the left panel, n = 4 for each group. ∗Significantly different; M.W.: molecular weight. (b) Immunoprecipitation of Ad-LacZ- and Ad-Cre-infected Ogtf/f and/or Ogtf/y neonatal cardiomyocytes using antibody against O-GLcNAc (RL2), followed by western blot using antibodies against O-GlcNAc (RL2) and ULK1. Ab.: antibody; M.W.: molecular weight. (c) Immunoprecipitation of Ad-LacZ- and Ad-Cre-infected Ogtf/f and/or Ogtf/y neonatal cardiomyocytes using antibody against ULK1, followed by western blot using antibodies against O-GlcNAc (RL2) and ULK1. Ab.: antibody; M.W.: molecular weight. N.S.: nonspecific band.

Fig 3: Prolonged inhibition of LRRK2 induces increase production of ULK1 and p62(A) Eighteen hours LRRK2in1 treatment to inhibit LRRK2 kinase. The gel shown is representative of three independent experiments, each of them performed with three replicates. The graph in (B) and (C) show quantification of (A). Ser758 ULK1 and total ULK1 were quantified against their own β-actin loading control; then, Ser758 ULK1 was normalized against total ULK1. Mean and standard deviation are shown, statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test. (D) Eighteen hours LRRK2in1 treatment to inhibit LRRK2 kinase or m-TOR inhibition by torin-1 in the presence and absence of bafilomycin-A1 to block autophagy. The gel shown is representative of three independent experiments. The graph in (E) shows quantification of (D). p62 was quantified against β-actin; mean and standard deviation are shown; statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test; **P<0.01 (F). Eighteen hours LRRK2in1 treatment to inhibit LRRK2 kinase in the presence and absence of cycloheximide to block protein synthesis. The gel shown is representative of three independent experiments, each of them performed with three replicates. p62 and total ULK1 were quantified against β-actin in (G) and (H) respectively. Mean and standard deviation are shown; statistical analysis was performed by ANOVA followed by Tukey’s post-hoc test; *P<0.05, **P<0.01 and ***P<0.001; ns = not significant.

Fig 4: Increased macroautophagy following inhibition of LRRK2 kinase is mTOR independent.(a) 150 minutes treatment with LRRK2in1 or Torin-1 (to induce macroautophagy through mTOR). Phosphorylation of P70S6K and ULK1 were used as readout for mTOR inhibition. (b) 150 minutes treatment with LRRK2in1 or Torin-1 in scramble controls and in H4 cells with stable ULK1 knock-down (~70% ULK1 knock-down). The gel shown is representative of 4 independent experiments that are quantified in (c). (c) Quantification was done for each single experiment and LC3-II was normalized against β-actin. After normalization against the control in DMSO, data were pulled together and statistical analysis was performed by 1way Anova followed by Tukey post-hoc test (mean and SD, *p < 0.05). (d) 18 hours treatment with LRRK2in1 in the presence and absence of Torin-1. Phosphorylation on P70S6K was used as control for mTOR inhibition. The gel shown is representative of 3 independent experiments each performed in triplicate and quantified in (e). (e) LC3-II was quantified against β-actin; statistical analysis was performed by 1way Anova followed by Tukey post-hoc test (mean and SD, **p < 0.01, ***p < 0.001).

Fig 5: Short term bed rest did not alter the protein content of regulators of Mitochondrial Biogenesis or Mitophagy No changes in skeletal muscle protein expression of p‐AMPKT172:total AMPKα1 (a), p‐ACCS79:total ACC (b), PGC1α (c), or TFAM (d), ULK1 (e), BNIP3L/Nix (f), or LC3BI/II (g) were observed pre‐ and post‐five days of bed rest with (EX) or without (CTRL) resistance exercise prehabilitation. Data presented as fold‐change from pre‐BR CTRL with mean ± SEM (n = 10 per group). Statistical significance was set at p < 0.05.