Fig 2: Generation and validation of the experimental model. e-AMPK WT/KO mice were intraperitoneally administered Tamoxifen (500 µg/mice) for 5 consecutive days in order to induce ?1AMPK invalidation specifically in the endothelium. 3 weeks after the last Tamoxifen injection, mice were sacrificed and endothelial cells were immunoprecipitated from lung tissue with dynabeads. ?1AMPK gene expression was detected by TaqMan qPCR on both isolated endothelial cells and supernatants. (a) Validation of endothelial ?1AMPK deletion’s extent in e-AMPK KO. ?1AMPK expression detected by TaqMan qPCR in immunoprecipitated endothelial cells. (b) Validation of endothelial ?1AMPK deletion’s specificity. ?1AMPK expression detected by TaqMan qPCR on lysates of lung tissue depleted of endothelial cells. (c) Time course of canagliflozin plasma levels in mice treated by oral gavage, 100 mg/kg, during indicated time. The data are mean ± SEM, n = 3 to 5/group. The data were analyzed using one-way ANOVA test.

Fig 3: ACSL4 regulated the AMPK/mTOR pathway in sev-stimulated SH-SY5Y cells. SH-SY5Y cells were transfected with si-ACSL4 or si-NC for 24 h and treated with 4.1% sev for 6 h, and then tested for the expression of AMPK, pAMPK, mTOR, and p-mTOR using western blot. (A) Representative images for western blot of AMPK, pAMPK, mTOR, and p-mTOR expression in SH-SY5Y cells are shown. Quantification of (B) p-AMPK/AMPK and (C) p-mTOR/mTOR ratios in SH-SY5Y cells. **, P<0.01; ***, P<0.001. ACSL4, acyl-CoA synthetase long chain family member 4; AMPK, 5' AMP-activated protein kinase; mTOR, mammalian target of rapamycin; sev, sevoflurane; pAMPK, phosphorylated AMPK; p-mTOR, phosphorylated mTOR; sev, sevoflurane.

Fig 4: AMPK activation by Canagliflozin protects VE-Cad organization and endothelial barrier function against LPS injury. (a) Schematic representation of the experimental protocol. For (b, c), HMECs were transfected with scramble or ?1AMPK targeting siRNA (50 nM) for 48 h, then treated with canagliflozin (Cana) (3 µM) or DMSO for one hour, before adding lipopolysaccharide (LPS) or vehicle (50 µg/mL) for 6 h. For (d), SBI compound was incubated for 15 min, then canagliflozin (3 µM) or DMSO were incubated for one hour, before adding LPS (50 µg/mL) for 6 h. (b, c) VE-Cad immunostainings were performed on HMECs treated according to the protocol detailed in (a). Representative images (b) and quantifications (c) are shown. Intercellular gaps are indicated by white arrows. Nuclei were stained with DAPI. Scale bar, 50 µm. (d) Endothelial permeability in response to canagliflozin, LPS challenge, and AMPK inhibitor SBI0206965. HMECs were grown on gelatin-coated Transwell inserts for 72 h and treated according to the protocol detailed in (a). Data are expressed as mean ± SEM (3 biological replicates for each condition). #p < 0.05 is relative to respective non-treated HMECs, $p < 0.05 is relative to LPS-only treated HMECs, and *p < 0.05 is relative to cells treated with DMSO. The data underwent two-way ANOVA.

Fig 5: Pistacia integerrima induces AMPK-dependent autophagic cell death in lung cancer cells. (A) Acridine orange staining of A549 cells post 24 h treatment with PI at indicated doses. Each image is zoomed in to clearly show the acidic vacuoles. The right panel shows the percentage of cells positive for acidic vesicular organelles (AO) in each treatment. Significance is evaluated with respect to untreated. (B) Whole cell lysates of A549 cells, post 24 h of treatment at indicated doses, were subjected to Western blotting using antibodies against LC3 A/B and pAMPK. ß-Actin served as a loading control. (B) Whole cell lysates of A549 cells treated at 10 µg/ml of PI for 6, 12, and 24 h time periods were subjected to Western blotting using antibodies against LC3 A/B and pAMPK. ß-Actin served as the loading control. (D) A549 cells treated with PI alone or together with chloroquine (CQ) for 24 h were stained with MDC. Fluorescence was expressed as fold change with respect to untreated. A significant difference in fold change of MDC staining is calculated in PI + CQ treatment with respect to PI. (E) Cell viability with Alamar blue was assessed in A549 cells treated with indicated treatments for 24 h. Cells were treated with chloroquine (CQ) at 10 µM and wortmannin (WTM) at 500 nM concentration. Significance in PI + CQ and PI + WTM is calculated with respect to PI alone. Error bars represent mean ± SEM; significance of data represented as *p < 0.05, **p < 0.005, ***p < 0.0005, and # p < 0.0001 and not significant (ns) if p > 0.05.