Fig 2: AMPK triggers post-translational modification of proteins involved in autophagy initiation. (A) Small luteal cells were pretreated with compound C (50 μM) for 1 h and then treated with AICAR (1 mM) for 15 min. Bar graphs and representative blots showing levels of phospho-AMPKα (Thr172), RPTOR (Ser792), ULK1 (Ser317), BECN1 (Ser93) and total BECN1. (B) Small luteal cells were infected with control adenovirus (Ad.CMV) or adenovirus expressing dominant negative AMPKα1 (Ad.dn.AMPKα1) for 48 h and then treated with AICAR (1 mM) for 15 min. Bar graphs and representative blots showing levels of phospho-RPTOR (Ser792), ULK1 (Ser317) and total AMPKα. Data are shown as a fold change and presented as means ± SEM from three-four separate experiments. Data were normalized to β-actin (ACTB). Statistical analysis was performed using one-way ANOVA test with post hoc Bonferroni test. Significant differences between treatments are indicated with asterisks * and ** reflecting p < 0.05 and p < 0.01.

Fig 3: Effect of CA-AMPK expression on synaptic integrity, density, and autophagic markers.Primary neurons were infected at 17 DIV with a constitutively active form of AMPK (CA-AMPK) for 7 days. b Cytotoxicity was assessed by measuring the lactate dehydrogenase (LDH) released in cell media. Treatment with 0.9% Triton X-100 was used as a positive control. Results represent mean ± SD, n = 4. Cells lysates were analyzed by WB (a) and the ratio pACC/ACC was quantified (c). d–i Quantification of the pre-synaptic markers SNAP25 (d), Munc (e), and synapsin Ia (f) and the post-synaptic markers PSD-95 (g), GluN1 (h), and GluA1 (i). Results represent mean ± SD, n = 8. j Immunostaining of synaptophysin (SYP, red) and PSD-95 (green) in primary neurons infected at 17 DIV with the CA-AMPK for 7 days. k Quantification of pre-synaptic (red), post-synaptic (green), and synaptic (yellow) puncta upon CA-AMPK expression. Results represent mean ± SEM, n = 38 neurons counted from five independent experiments. Scale bar = 50 µm. l–p Analysis of the expression of proteins involved in autophagy pathway. Quantification of the ratios pRaptor/Raptor (m) and pULK/ULK (n) and of the expression level of p62 (o) and LC3 II proteins (p). Results represent mean ± SD, n = 6. Student’s t-test was performed for c–i and m–p experiments. One-way ANOVA with Bonferroni’s post hoc test was used for b, k experiments; *p < 0.05, **p < 0.01, ***p < 0.001 compared to Ctrl

Fig 4: Inhibition of cellular glucose metabolism blocks WST-8 reduction. (A~D) Increasing amount of inhibitors was used to treat HEK293 cells for 6 hours. Absorbance of WST-8 formazan was recorded after incubation for 1 hour. Data of GLUT inhibitor II (A), hexokinase inhibitor 2-DG (B), GAPDH inhibitor Iodoacetate (C) and G6PD inhibitor (DHEA) (D) were presented. (E) HEK293 cells were treated with inhibitors including GLUT inhibitor II (25μM), 2-DG (25mM), DHEA (62.5μM) for 6 hours. Phosphorylation of ACC on serine-79, Raptor on serine-792, S6K1 on threonine-389, Akt on threonine-308 and expression of protein interacting with NIMA (Pin1), beta-tubulin or GAPDH were determined by Western Blot.

Fig 5: WST-8 assay identifies ritonavir as an inhibitor of cellular glucose metabolism. (A) HEK293 cells were treated with increasing amount of clozapine, dexamethasone, hydrochlorothiazide or ritonavir for 6 hours. Absorbance of WST-8 Formazan was recorded after incubation for 1 hour. (B) HEK293 cells were treated with increasing doses of ritonavir for 6 hours. Absorbance of WST-8 Formazan was recorded after incubation for 1 hour. Intracellular ATP was measured by ATP assay. Relative WST-8 Formazan absorbance or ATP was normalized to its parallel controls. Data of three independent replicates are presented as the mean+/-s.e.m., n=3. (C) HEK293 cells were treated with increasing doses of ritonavir. Phosphorylation of ACC on serine-79, Raptor on serine-792, S6K1 on threonine-389, and expression of Actin were determined by Western Blot.