Variance stabilization of Affymetrix oligo-array dataset by means of VSN arcsinh transformation () or spline fit ()

<p><b>Copyright information:</b></p><p>Taken from "An adaptation of the LMS method to determine expression variations in profiling data"</p><p></p><p>Nucleic Acids Research 2007;35(9):e71-e71.</p><p>Published online 25 Apr 2007</p><p>PMCID:PMC1888829.</p><p>© 2007 The Author(s)</p> The SD values (-axis) are plotted against a running index (-axis) where each value corresponds to a gene, from 6070 randomly selected genes. Red dots indicate the median of SD calculated on bins each corresponding to 10% of ranked genes

Differentially expressed genes selected on spline (EV) or VSN transformed data

<p><b>Copyright information:</b></p><p>Taken from "An adaptation of the LMS method to determine expression variations in profiling data"</p><p></p><p>Nucleic Acids Research 2007;35(9):e71-e71.</p><p>Published online 25 Apr 2007</p><p>PMCID:PMC1888829.</p><p>© 2007 The Author(s)</p> The same dataset represented in was used to select differentially expressed genes on spline (EV) or VSN-transformed data. MA plots of scaled log-transformed signals for probe sets from two experimental conditions of the B6.1 cells. Color codes are as in . Up-regulated genes are shown as green bold dots, down-regulated as red bold dots. () Distribution of 555 genes (-value = 0.05) selected upon a spline fit. () Distribution of 555 genes (FC = 1.63) on VSN-transformed data. () Distribution of genes selected by both approaches. () Distribution of genes selected by EV (spline fit) alone. () genes selected by VSN (arcsinh transformation) alone

Gene expression scatter plot of Affymetrix oligo-array data and EV unit

<p><b>Copyright information:</b></p><p>Taken from "An adaptation of the LMS method to determine expression variations in profiling data"</p><p></p><p>Nucleic Acids Research 2007;35(9):e71-e71.</p><p>Published online 25 Apr 2007</p><p>PMCID:PMC1888829.</p><p>© 2007 The Author(s)</p> () Dataset generated with Affymetrix chips. Hybridization, washes, antibody amplification and staining were performed using the Affymetrix fluidics station and scanner following the manufacturer's instructions. Data was log scaled; scatter plots show uneven variance resulting in increased dispersion at low expression levels. Note that the first diagonal (black) and the median axis (yellow) do not overlap. Shown on the plot Median and confidence interval curves corresponding to parameter setting of α = 0.317 (green upper limit, red lower limit) are also shown on the plot. () DS (α = 0.317, green and red plain curves) and iso-variation representing expression variation EVα(X) = 2 (green and red dotted curves) correspond to the confidence limits defined with a -value = 0.05. For each spot S, its orthogonal projection to the median O was determined and the distance between O and U (O,U) (intersection of the segments O,U and the curve of the DS) measured. For each spot located along the O, S line, the value (O,U) represents the unit of expression. Expression variation Evα(S) thus corresponds to the (O,S)/(O,U) ratio

Differentially expressed genes selected using spline or MAS5 Affymetrix selection

<p><b>Copyright information:</b></p><p>Taken from "An adaptation of the LMS method to determine expression variations in profiling data"</p><p></p><p>Nucleic Acids Research 2007;35(9):e71-e71.</p><p>Published online 25 Apr 2007</p><p>PMCID:PMC1888829.</p><p>© 2007 The Author(s)</p> MA plots of scaled log-transformed signals for probe sets from two experimental conditions of the B6.1 cells, using the same dataset and color codes as in . () Distribution of 555 genes (-value = 0.05) selected upon a spline fit. () Distribution of 555 genes selected by MAS5 (FC = 1.8). () Distribution of genes selected by both approaches. () Distribution of genes selected by EV (spline fit) alone. () Genes selected by FC on MAS5 alone

Melatonin does not inhibit aerobic glycolysis in chondrosarcoma cells.

<p>(A) Glucose uptake was determined in sw-1353 chondrosarcoma cells treated with o without 1 mM melatonin for several times (2h, 4h, 6h and 8h). Data are expressed as RFU (relative fluorescence units). (B) Melatonin effects on glycogen stores were evaluated in sw-1353 cells incubated with 1 mM melatonin for 24 hours, by means of electron microscopy. Black arrows indicate glycogen stores that appear at cytoplasm forming rosettes. Also dense granules of glycogen can be observed free in the cytoplasm in both control and melatonin treated groups. N, nucleus; m, mitochondria; G, golgi apparatus areas. Bars: 0.5μm. (C) Intracellular glycogen levels were assessed in sw-1353 cells after incubation with 1 mM melatonin for 4 and 24 hours. (D) Chondrosarcoma cells were treated with or without 1 mM melatonin and 10 μM CP316819 for 72 hours, and cell number was determined by the quantification of total LDH present in cell culture. Data are expressed as the percentage of control (vehicle-treated cells). *p≤0.05 vs. vehicle-treated cells.</p

Ewing sarcoma cells exhibit the main hallmarks of Warburg effect.

<p>The following parameters indicative of aerobic glycolysis were determined in TC-71 and sw-1353 cell lines (*p≤0.05 vs. sw-1353 cell line, unless otherwise indicated): (A) Glucose uptake (relative fluorescence units, RFU); (B) Intracellular lactate levels (nmoles/μl); (C) LDH activity, normalized versus protein content; (D) ATP levels (millimoles of ATP/μg protein); and (E) Mitochondrial membrane potential (ΔΨm) was expressed as RFUs. (F) TC-71 cells were treated with or without 16.2 mM oxamate for 48 hours, and cell survival rate was determined by means of trypan blue staining. Results were expressed as a percentage of death cells. *p≤0.05 vs. vehicle-treated cells.</p

Aerobic glycolisis is inhibited by melatonin in Ewing sarcoma cells.

<p>(A) Lactate levels (nmoles/μl) were quantified in TC-71 cells treated with 1 mM melatonin for 2, 4, 6 and 24 hours. (B) LDH activity was evaluated in TC-71 cells incubated with 1 mM melatonin for several times (2h, 4h, 6h and 24 hours), and results were normalized with protein content (μg) in each sample. (C) Intracellular lactate levels and LDH activity were evaluated in A-4573 and A-673 Ewing sarcoma cells treated with 1 mM melatonin for 24 hours. Data are represented as percentage versus control group. (D) Cell viability (% of death cells) was evaluated by trypan blue after the incubation of TC-71, A4573 and A-673 Ewing sarcoma cells with 1mM melatonin and 16.2 mM oxamate for 48 hours. (E) Intracellular lactate levels (nmoles/μl) were quantified in sw-1353 chondrosarcoma cells treated with 1 mM melatonin for 2, 4, 6 and 24 hours. (F) LDH activity was evaluated in sw-1353 chondrosarcoma cells incubated with 1 mM melatonin for 2, 4, 6 and 24 hours, and results were normalized with protein content (μg) in each sample. *p≤0.05 vs. vehicle-treated cells; #p≤0.05 vs. melatonin-treated cells.</p

Melatonin inhibits HIF-1α in a PI3K/AKT/mTOR independent pathway.

<p>Western blot analyses were carried out to identify the effect of melatonin (1 mM, 4 and 8 hours) on the activation of HIF-1α in TC-71 cells (A) and A-4573 and A-673 (1mM, 4 hours) cells (B). AKT and mTOR activation in TC-71 (C) and A-4573 and A-673 cells (D) was evaluated using specific phosphoantibodies. GAPDH was used as a loading control in all cases. A representative blot is showed. Optical density of bands was measured and values of the hydroxy-HIF-1α (inactivated form), p-AKT or p-mTOR bands were normalized versus GAPDH. Results are represented as percentage of the values found in vehicle-treated cells (dotted line). (E) <i>Left panel</i>, cell viability was evaluated by MTT reduction assay after treatment of TC-71 cells with 1 mM melatonin alone or in combination with 10 nM rapamycin or 10 μM LY294002 for 48 hours. Data are expressed as the percentage of vehicle-treated cells. <i>Right panel</i>, Representative western blot showing the relative protein level of p-AKT, p-mTOR and hydroxy-HIF-1α after 10 nM rapamycin or 10 μM LY294002 treatment during 24 hours in TC-71 cell line. p*≤0.05 vs. vehicle-treated cells.</p

Melatonin inhibits glucose uptake and depletes glycogen stores in Ewing sarcoma cells.

<p>(A) Glucose uptake was measured in TC-71 cells treated with o without 1 mM melatonin for several times (2h, 4h, 6h and 8h). Data are expressed as RFU (relative fluorescence units). (B) Glucose uptake was measured in A4573 and A-673 cells treated with o without 1 mM melatonin for 8h. Data are expressed as RFU (relative fluorescence units). (C) Electron microscopy images showing the decrease in glycogen stores after treatment of Ewing sarcoma cells with 1 mM melatonin for 24 hours. Black arrows indicate glycogen stores that appear at cytoplasm forming rosettes. Also dense granules of glycogen can be observed free in the cytoplasm in control groups. N, nucleus; m, mitochondria; G, golgi apparatus areas. Right images on each experimental group correspond to a higher magnification of left image. Bars: 1μm (left image) /0.5μm (right image). (D) Intracellular glycogen levels were assessed in TC-71, A-4573 and A-673 cells after incubation with 1 mM melatonin for 4 hours. (E) Cell death was determined by means of the LDH-release assay. Ewing sarcoma cells were treated with or without 1 mM melatonin and 10 μM CP316819 for 72 hours, and cell death was calculated as the ratio between released and total LDH activity on each experimental group. Data are expressed as the percentage of control (vehicle-treated cells).*p≤0.05 vs. vehicle-treated cells; #p≤0.05 vs. melatonin-treated cells.</p

Additional file 4: of Class-modeling analysis reveals T-cell homeostasis disturbances involved in loss of immune control in elite controllers

Partial least-squares–class modeling (PLS-CM). (DOC 33 kb