Fig 2: Melatonin acts differentially in tumoral and normal placental cells.(a) In the BeWo choriocarcinoma cell line, melatonin increases the phosphorylation of 5 'adenosine monophosphate-activated protein kinase (AMPK), due to the reduction of ATP levels [28]. Melatonin inhibits autophagic activity as well as the activation of the nuclear factor (erythroid-derived 2) -like 2 (Nrf2) transcription factor, contributing to BeWo cell death. (b) In primary cell cultures of human placental villous cytotrophobasts (vCTBs), H/R disrupts the cellular homeostasis. Melatonin treatment induces activation of two pro-survival mechanisms: autophagy and Nrf2, which help to restore homeostasis and cell survival.

Fig 3: Metformin reduces EOC cell growth in an AMP-activated protein kinase (AMPK)-independent manner. (A) Epithelial ovarian cancer (EOC) cell lines that stably express sh-control or sh-TOM40 were harvested at 70% confluency. The expression levels of indicated proteins were measured by western blot analysis. (B) sh-TOM40 stably expressed EOC cells were introduced to si-Luciferase or si-AMPKa1 and then incubated in a CO2 incubator (5% CO2 and 37 ?) for 72 h. Cell viability was assessed by crystal violet assay (Left panel). Data in the bar graph are expressed as the mean of absorbance ± Standard Deviation (S.D.)., n = 16. # p > 0.05. The expression levels of indicated proteins including anti-AMPKa1 were measured by western blot analysis (Right panel). (C) Cell viability (relative absorbance at 550 nm) as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolum bromide (MTT) assay. OVCAR-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 1 mM AICAR, 5 mM metformin, or 1 mM aspirin for 48 h. AICAR, 5-aminoimidazole-4-carboxamid ribonucleotide. (D) SK-OV-3 that stably express an empty vector or TOM40 was cultured with or without 25 mM glucose and with or without 5 mM metformin for 48 h. Results are shown as the relative means ± S.D., n = 6; # p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. (E) Western blot analysis of proteins expressed by EOC cell lines, OVCAR-3 and SK-OV-3, that stably express empty vectors or TOM40 and were cultured in dulbecco modified eagle medium (DMEM) with or without 25mM glucose and with or without 1 mM AICAR or 5 mM metformin for 24 h.

Fig 4: TSPAN1 increases cell growth through AMPK activity in the endometriosis cell line. (A) Protein levels were detected by immunoblotting in stable cell lines, with a-actinin as an internal loading control (B) Protein levels were detected by immunoblotting after treatment of inhibitors in 6045_SV40 stable cell lines. (DMSO: control; 500 nm wortmannin: PI3 kinase inhibitor; 5 µm U0126: ERK inhibitor; 1 µm Compound C: AMPK inhibitor), with a-actinin as an internal loading control. (C) Cell proliferation was determined via WST-1 after treatment of inhibitors in 6045_SV40 stable cell lines. Unpaired t-test was performed. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Cell invasion analysis of stable cell lines using a Matrigel invasion assay after treating 6045_SV40 stable cells with inhibitors. Upper panel shows quantification of invasion experiments (200× magnification); lower panel shows representative images. Results are presented as the relative number of invading cells in five randomly selected fields. Error bars represent mean ± standard error (SE); n = 3. Unpaired t-test was performed. *P < 0.05, ***P < 0.001. (E) Protein levels were confirmed by immunoblotting after treatment of DMSO and 0.5 and 1 µm of Compound C in 6045_SV40 stable cell lines, with a-actinin as an internal loading control. (F) Cell proliferation was measured via crystal violet assay after treatment of DMSO and 0.5 and 1 µm of Compound C in 6045_SV40 stable cells. Upper panel shows representative images; lower panel shows the relative absorbance at 595 nm. Error bars represent mean ± SE; n = 3 Unpaired t-test was performed. *P < 0.05, **P < 0.01. (G) Protein expressions were detected by immunoblotting after transient transfection of siControl or siAMPK alpha1 in 6045_SV40 stable cells, with a-actinin as an internal loading control. (H) Cell proliferation was measured via crystal violet assay after transient transfection of siControl or siAMPK alpha1 in 6045_SV40 stable cells. Upper panel shows representative images; lower panel shows the relative absorbance at 595 nm. Data are expressed as the mean ± SE; n = 3. Unpaired t-test was performed. ***P < 0.001.

Fig 5: Effects of EMPA on AMPK/SIRT1 and oxidative stress in cultured human podocytes. (A) Protein levels of SIRT1, AMPK, p-AMPK, Foxo1 and SOD2 in podocytes determined by Western blot analysis. (B) Densitometric evaluation of Western blots for SIRT1 (n = 3), (C) p-AMPK (n = 3), (D) Foxo1 (n = 3) and (E) SOD2 (n = 3). (F) Representative immunofluorescence images of SIRT1, p-AMPK, Foxo1 and SOD2 in podocytes. Scale bar: 100 µm. Data are expressed as the mean ± SEM and were analyzed by ANOVA with LSD post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.