Three gene combinations to predict the behavior of selected target genes

<p><b>Copyright information:</b></p><p>Taken from "Insight into redox-regulated gene networks in vascular cells"</p><p>Bioinformation 2007;1(10):379-383.</p><p>Published online 10 Apr 2007</p><p>PMCID:PMC1896051.</p><p></p> The values are the COD values for each predictor and the effect that addition of each predictor has on overall model prediction potentia

(A) Soft agar colony formation assays were performed on each clone as a measure of cell-adhesion growth. IH-selected clones demonstrated an increase in the number of colonies relative to passage control clones. Metastatic MDA-MB-231 cells were seeded as a positive control. (B) Passage control MCF01A clones (top) and IH-selected clones (bottom) were imaged using brightfield microscopy. While control clones exhibited smooth borders indicative of an epithelial phenotype, IH-selected cells migrated apart from adjacent cells, indicating an increase in migration as well as EMT. (C) As a measure of migratory capacity, passage control MCF10A clones (in grey) and IH-selected MCF10A (in black) were seeded within an xCELLIGENCE CIM plate, which measures in real time the number of cells passing through a barrier from a low-serum well to high-serum well. Measurements taken over the span of 24 hours indicates IH-selected clones have increased migratory rates (D) Proliferation rates of each of the fo

<p>(A) Soft agar colony formation assays were performed on each clone as a measure of cell-adhesion growth. IH-selected clones demonstrated an increase in the number of colonies relative to passage control clones. Metastatic MDA-MB-231 cells were seeded as a positive control. (B) Passage control MCF01A clones (top) and IH-selected clones (bottom) were imaged using brightfield microscopy. While control clones exhibited smooth borders indicative of an epithelial phenotype, IH-selected cells migrated apart from adjacent cells, indicating an increase in migration as well as EMT. (C) As a measure of migratory capacity, passage control MCF10A clones (in grey) and IH-selected MCF10A (in black) were seeded within an xCELLIGENCE CIM plate, which measures in real time the number of cells passing through a barrier from a low-serum well to high-serum well. Measurements taken over the span of 24 hours indicates IH-selected clones have increased migratory rates (D) Proliferation rates of each of the four clones. Cell counts were performed daily. IH-selected clones exhibited reduced proliferation relative to passage control clones. (E) Oxygen consumption and proton production (a proxy for glycolysis) were measured for each clone with the Seahorse XF Analyzer. The ratio of oxygen consumption to proton production was increased in IH-selected clones.</p

SU86.86 cells exhibit phenotypic and genotypic changes following selection by IH.

<p>SU86.86 cells were treated with IH for 50 cycles, had clonal populations raised, and grown in normoxia for 2 months. A significant decrease in expression of E-cadherin (A) and p53 (B) was detected by quantitative Real-Time PCR. IH-selected SU86.86 clones also exhibited an increase in resistance to etoposide (C) and hypoxia (D).Results of selection of RKO cells following IH-selection (E-H). We do not detect any changes in p53 or E-cadherin expression within clones of RKO IH-selected (E,F). Furthermore, these cells did not exhibit resistance to etoposide (G), nor hypoxia (H).</p

CA-IX is expressed in pseudohypoxic tissue.

<p>200x magnification image of ductal carcinoma of the breast with microinvasion A) stained against CA-IX and B) segmented into individual cells and color coded by quantitative intensity where white is excluded as stroma, yellow is weak, orange is moderate and red is strong staining intensity. C) Bar graph of CA-IX intensity per lesion type from low grade DCIS to high grade DCIS and DCIS with microinvasion.</p

Selection of MCF10A cells by intermittent hypoxia (IH) induces drug resistance and reduced expression of p53 and E-cadherin.

<p>(A) Multiple intermittent hypoxia regimens were tested at 1%O₂ and 0.2% O₂ over 6 days. Factor survival is expressed as compared to cells grown in parallel in normoxia (21%O₂). MCF10A cells undergoing repetitive cycles of 16 hours hypoxia followed by 8 hrs reoxygenation exhibited the most cell death. (B) MCF10A cells were cultured in 50 cycles of IH in the selective regimen described in (A), and individual cells were isolated to be raised as clones, and then passaged for 2 months in normoxia. These clonal populations were tested for etoposide resistance. Heterogeneity in the IH-selected clones was detected, while a significant overall increase in etoposide resistance was measured. (C) MCF10A IH-selected clones 4 and 9 were further tested for resistance to multiple cytotoxic conditions, including treatment to the microtubule stabilizing docetaxel, the folate metabolism inhibitor methotrexate, as well as hypoxia and reduction of growth factors. Intermittent hypoxia exhibited increased survival to all of these conditions except for survival to growth factor removal. (D,E) Intermittent hypoxia exhibits changes in the expression of p53 and E-cadherin. Quantitative Real-Time PCR was performed on each IH-selected and passage control clone. Reduced <i>p53</i> and E-cadherin mRNA expression levels were detected in IH-selected control clones. Factor expression is relative to the average of control clones.</p

Details of the significant protein markers with FDR and fold change (log of Control to IH-selected group).

<p>Details of the significant protein markers with FDR and fold change (log of Control to IH-selected group).</p

Differential protein levels and phenotypes are exhibited among MCF10A cells following selection through IH.

<p>(A) Analysis of protein levels was performed using the Reverse Phase Protein Array (RPPA) on the parental MCF10A line, passaged control clone 7, IH-selected clone 4, and IH-selected clone 9. Each clone was submitted in duplicate. Hierarchical clustering of significant (FDR ≤ 0.05) protein markers in control versus IH-selected samples is shown. The clustering was carried out with Euclidean distance metric with average linkage, and the data was median normalized and log2 transformed. The clustering was arbitrarily stopped at 2 and 5 groups for the samples and features respectively. (B) Western blotting analysis of p53 and E-cadherin protein. p53 protein is reduced by in IH-selected clones. E-cadherin is completely lost following selection by IH, and N-cadherin protein is detectable, indicating EMT in IH-selected clones. (C) Quantitative RT-PCR was performed on DNA levels of the <i>P53</i> and <i>E-CAD</i> loci. A 0.5 fold reduction in the number of copies of <i>P53</i> and <i>E-CAD</i> indicates a loss of one allele of each. (D) Western blotting against HIF-1α and GLUT-1 indicates constitutive stabilization of HIF-1α and its downstream effector GLUT-1 in IH-selected cells. MCF10A cells cultured in hypoxia are used as a positive control.</p

Multivariable Cox Proportional Hazards Models for Progression Free and Overall Survival for the Screen-Detected Cohorts.

<p>Multivariable Cox Proportional Hazards Models for Progression Free and Overall Survival for the Screen-Detected Cohorts.</p

Baseline Demographics and Clinical Characteristics of Four Screen-Detected Lung Cancer Case Cohorts.

<p>Baseline Demographics and Clinical Characteristics of Four Screen-Detected Lung Cancer Case Cohorts.</p

Schema for the lung cancer case cohorts in the NLST at prevalence (baseline) and incidence screening (follow-up) rounds.

<p>Abbreviations: T0 = baseline screen; T1 = first screen; T2 = second screen; [+] = positive screen; [–] = negative screen. Prevalence lung cancers diagnosed at T0 are shaded green. Screen-detected lung cancers diagnosed at T1 and T2 rounds in which T0 screens were positive are shaded blue. Screen-detected lung cancers diagnosed at T1 and T2 in which T0 screens were negative are shaded red. Interval cancers (beige boxes) were diagnosed following a negative screen. Participants were excluded if their screening results were inadequate or not compliant. At baseline we excluded 413 screens, 1,471 screens at T1, and 713 screens at T2.</p