Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced alterations in mRNA and protein expression of G2 checkpoint regulators. A. Gene expression of positive G2 checkpoint regulators in U2OS cells. The ratio of mRNA expression in cells treated with hypoxia (0.2% O2, 24 h) relative to mRNA expression in cells cultured at normoxia (21% O2) is shown. Data were obtained from genome wide microarray analysis. The positive G2 checkpoint regulators were found from published studies as described in Table 1. B. Gene expression of negative G2 checkpoint regulators similar as in A. C. Immunoblot analysis of protein extracts from U2OS cells exposed to hypoxia or normoxia for 24 h. The samples are from the same experiment as the microarray results shown in A and B. HIF1α was shown to confirm hypoxia. H4 was used as loading control.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Gene Expression, Cell Culture, Genome Wide, Microarray, Western Blot, Control

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Protein levels of G2 checkpoint regulators in individual G2 cells following hypoxia. A. Cell cycle profiles of U2OS cells after hypoxia treatment as in Figure 1 (24 h 0.2% O2). Flow cytometric analysis was performed after staining with anti phospho‐H3Ser10 (H3P) to mark mitotic cells, and the DNA stain Hoechst. Numbers indicate fraction of mitotic cells. B. Flow cytometric barcoding analysis for accurate measurement of protein levels in G2 phase cells. U2OS cells treated with four different conditions, as indicated in the right column, were labeled with different concentrations of Pacific Blue and combined into a single sample. The single sample of cells was then stained with antibodies to Cyclin B and phospho‐H3 and with the DNA‐stain FxCycle Far Red, and analyzed by flow cytometry. Gating of the Pacific Blue‐SSC plot (left) was used to separate the four original samples. The G1, S, G2 and M cell cycle phase populations were gated from the scatter plot of phospho‐Histone H3(Ser10) (H3P) versus DNA content, and the median signal for Cyclin B levels in each cell cycle phase could thus be obtained. C.Median values of G2 phase levels of the indicated proteins obtained as in B, after subtraction of background values obtained as in Figure S1. U2OS cells were grown at 21% O2, or exposed to 24 h hypoxia at 0.2% O2, or first exposed to 0.2% O2 for 24 h followed by subsequent incubation at 21% O2 for 90 min (90 min reox) or 6 h (6 h reox). Average results from at least 3 independent experiments are shown. Error bars indicate SEM.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Staining, Labeling, Flow Cytometry, Incubation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: Hypoxia‐induced changes in CDK activity and G2 checkpoint activation. A. CDK activity in G2 phase cells as measured by phosphorylation of BRCA2‐Ser3291. U2OS cells were grown at 21% O2, or incubated at 0.2% O2 for 24 h and harvested inside the hypoxia chamber and at 1 and 4 h after reoxygenation, or treated with Roscovitine for 2 h at 21% O2. Flow cytometry barcoding analysis of phospho‐BRCA2‐Ser3291 was performed as in Figure 2 and S2. B. Immunoblot analysis of U2OS cells treated as in A with antibodies to total BRCA2 and γ‐tubulin (loading control). C. G2 checkpoint activation after IR (0.2%O2 24 h). Flow cytometric analysis of G2 checkpoint arrest after X‐ray irradiation (0, 0.5, 1 Gy) of normoxic U2OS cells (21%O2) or U2OS cells exposed to 24 h of hypoxia at 0.2%O2 and irradiated 15 min after reoxygenation. Nocodazole was added to all samples 1 h after IR, and the samples were harvested 5 h later. The relative mitotic fraction was determined as the fraction of phospho‐H3 positive cells in irradiated samples divided by the fraction of phospho‐H3 positive cells in non‐irradiated samples. Average values from 3 independent experiments are shown. Error bars indicate SEM. D. Phosphorylation of BRCA2‐Ser3291 in H460 cells treated with hypoxia and analyzed as in A. E. G2 checkpoint activation in H460 cells treated with hypoxia and IR and analyzed as in C.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Activity Assay, Activation Assay, Phospho-proteomics, Incubation, Flow Cytometry, Western Blot, Control, Irradiation

Journal: Molecular Oncology

Article Title: Hypoxia‐induced alterations of G2 checkpoint regulators

doi: 10.1016/j.molonc.2015.12.015

Figure Lengend Snippet: IR‐induced G2 checkpoint and expression of G2 checkpoint regulators in U2OS cells after severe hypoxia (∼0.03% O2 20 h) and prolonged mild hypoxia (0.2%O2, 72 h). A. Similar G2 checkpoint measurement after IR as in Figure 3C following incubation at severe hypoxia (∼0.03% O2 20 h). B. Similar as in A following incubation at prolonged mild hypoxia (0.2% O2 72 h). C. Similar flow cytometric barcoding analysis of protein levels in G2 phase cells as in Figure 2C following incubation at severe hypoxia (∼0.03% O2 20 h). D. Similar as in C following incubation at 0.2% O2, 72 h.

Article Snippet: Human U2OS osteosarcoma, HeLa cervical carcinoma cells and NCI–H460 lung cancer cells (ATCC) were cultured in DMEM (Dulbecco's modified Eagle's) medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (P/S) at 37 °C in a humidified atmosphere with 5% CO 2 .

Techniques: Expressing, Incubation