Additional file 2: Figure S1. of Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells

Clusterization of CC enriched after T3 treatment in HeLa and MCF-7 cells. The distances between enriched CC were estimated according to the Resnik measure. The optimal number of clusters was estimated by silhouette scores and represented with different colors within the dendrogram. The box on the right side of the figure shows CCs enriched by a specific treatment. (TIF 3584 kb

Additional file 4: Figure S3. of Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells

Modulation of MIC-1 mRNA expression. MIC-1 mRNA upregulation induced by T3 was partially prevented by pre-treatment with ICI-182,780. Once synchronized, 300.000 cells were seeded onto multi-well plates and pre-incubated with ICI-182.780 (10−5 M) for 30 min. TRF, purified T3 or α-TOC were added to the culture medium for 24 h. Gene expression was analyzed by real-time quantitative PCR, and results were log transformed (logarithm 2) in order to obtain data symmetrically distributed [46]. Statistical significance was calculated by one-way ANOVA with repeated measures followed by Tukey’s test using ΔCt value. Asterisks (*) indicate significant differences (p value ≤0.05) with respect to control. Data represent the pooled values of at least three independent experiments. (TIF 15116 kb

Additional file 3: Figure S2. of Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells

Densitometric analysis of ATF-6, PERK, and IRE-1α activity. Treatments with T3 have no significant effect on the protein expression of ATF-6 and PERK at 24 h from the treatment. Conversely, IRE-1 phosphorylation is significantly affected by T3 treatment. Data were analyzed by one-way ANOVA with repeated measures followed by Fisher’s test. Asterisks indicate significant differences (p value ≤0.05) between treated cells vs control (CC). (TIF 13186 kb

Additional file 1: Table S1. of Tocotrienols induce endoplasmic reticulum stress and apoptosis in cervical cancer cells

List of genes modulated by T3 according to microarray analysis. Expression levels are represented as log2 fold changes. The last column on the right indicates if the gene has been reported to be involved in EndoR stress. (DOCX 49 kb

Nanomolar Caffeic Acid Decreases Glucose Uptake and the Effects of High Glucose in Endothelial Cells

<div><p>Epidemiological studies suggest that moderate and prolonged consumption of coffee is associated with a reduced risk of developing type 2 diabetes but the molecular mechanisms underlying this effect are not known. In this study, we report the effects of physiological concentrations of caffeic acid, easily achievable by normal dietary habits, in endothelial cells cultured in 25 mM of glucose (high glucose, HG). In HG, the presence of 10 nM caffeic acid was associated with a decrease of glucose uptake but not to changes of GLUT-1 membrane localization or mRNA levels. Moreover, caffeic acid countered HG-induced loss of barrier integrity, reducing actin rearrangement and FITC-dextran passage. The decreased flux of glucose associated to caffeic acid affected HG induced apoptosis by down-regulating the expression of initiator (caspase 8 and 9) and effector caspases (caspase 7 and 3) and by increasing the levels of phosphorylated Bcl-2. We also observed that caffeic acid in HG condition was associated to a reduction of p65 subunit nuclear levels with respect to HG alone. NF-κB activation has been shown to lead to apoptosis in HG treated cells and the analysis of the expression of a panel of about 90 genes related to NF-κB signaling pathway revealed that caffeic acid significantly influenced gene expression changes induced by HG. In conclusion, our results suggest that caffeic acid, decreasing the metabolic stress induced by HG, allows the activation of survival mechanisms mediated by a different modulation of NF-κB-related signaling pathways and to the activation of anti-apoptotic proteins.</p></div

Mapping of the significant modulated genes within Reactome pathways database tool.

<p>The color of the nodes represents the FC expression associates to HG, the color of the frame of the nodes corresponds to the FC expression associated to AC+HG. The color intensity of the nodes is associated to the FC rate as indicated in the figure.</p

Analysis of activated caspases and anti-apoptotic Bcl-2.

<p>A) A representative Western blot of procaspase and cleaved caspase-8 after 12 hours of treatments. B) A representative Western blot of caspase-7 and procaspase-9 after 12 and 24 hours of treatments. C) A representative Western blot of procaspase and cleaved caspase-3 after 12 hours of treatments. Caspase 3 activity was measured by detection of fluorescence of the caspase substrate after 12, 24 and 36 hours of treatments. Data are expressed as relative AFC (7-amino-4-trifluoromethylcpumarin) fluorescence normalized for total protein content in each sample. D) Effect of CA on phosphorylated Bcl-2 protein levels at 12 and 24 hours of treatments. Images report one representative experiment out of at least 3 independent experiments.*p<0.05 <i>vs</i> control; **p<0.001 <i>vs</i> control; # p<0.05 CA+HG <i>vs</i> HG.</p

CA protects HG-dependent loss of endothelial barrier integrity.

<p>Cells were seeded onto PET Transwell filters and incubated in control, CA, HG and CA+ HG conditions for 24, 30, 48 and 72 hours. FITC-FD40 was added to a final concentration of 500 μg/ml in the apical medium. The fluorescence was measured both in basal and apical medium. Data are presented as percentage of the ratios of FD40 basal <i>vs</i> apical fluorescence. ***p<0.0001 <i>vs</i> control; n.d. = not detected. Values are the mean ± SEM from at least 3 independent experiments.</p

Effect of CA on cell proliferation and apoptosis.

<p>Cells were treated for 24, 48 and 72 hours as indicated in the figures. Panels show: A) cell count expressed as number of cells per well; B) Proliferation analysis by BrdU incorporation; C) Representative dot plots of cell distribution after double labeling with Annexin-V and PI. Data are expressed as percentage of induction of early (Annexin V positive cells) and late stages of apoptosis (Annexin-V/PI double positive cells). *p<0.05 HG <i>vs</i> control; **p<0.001 <i>vs</i> control; # p<0.05 CA+HG <i>vs</i> HG. ## p< 0.001 and ### p< 0.0001 CA+HG <i>vs</i> HG. Values are the mean ± SEM from at least 3 independent experiments.</p

CA reduces HG-dependent actin reassessment.

<p>Cells were seeded onto PET Transwell filters and incubated in control, HG or CA+HG conditions for 48 hours. Cells were fixed in 2% PFA and permeabilized with 0.1% Triton X-100. F-actin filaments and nuclei were stained with FITC-conjugated phalloidin (green) and DAPI (blue), respectively. White arrows indicate peripheral F-actin. The image reports one out of at least 3 independent experiments.</p