Journal: Stem cells (Dayton, Ohio)

Article Title: MEK-ERK signaling dictates DNA-repair gene MGMT expression and temozolomide resistance of stem-like glioblastoma cells via the MDM2-p53 axis.

doi: 10.1002/stem.753

Figure Lengend Snippet: Figure 1. MGMT expression and temozolomide resistance of stem-like glioblastoma cells. (A): MGMT expression levels in the indicated stem- like glioblastoma cells were determined by immunoblot analysis. (B): The stem-like glioblastoma cells were exposed to TMZ at the indicated con- centrations for 4 hours, and the percentage of dead cells was determined after 72 hours. The data represent the means þ standard deviations of three independent experiments. (C, D, E): The stem-like glioblastoma cells were transfected with the indicated siRNAs against MGMT or with a nontar- geting control (Cont.). The cells were then subjected to immunoblot analysis for MGMT expression (C) or to cell death assay using 50 lM temozo- lomide (D and E) 72 hours after transfection. In (D), representative images of cell death assay are shown, where the nuclei of propidium iodide- positive, dead cells are stained red while those of live cells are stained blue with Hoechst 33342 (scale bar ¼ 200 lm). In (E), the graph shows the percentage of dead cells (means þ standard deviations of three independent experiments). *, p < .01, **, p < .05, n.s., not significant. Abbrevia- tions: MGMT, O6-methylguanine DNA methyltransferase; siRNA, short-interfering RNA; TMZ, temozolomide.

Article Snippet: SL327 was purchased from Enzo Life Sciences (New York, NY, http://www.enzolifesciences.com), temozolomide was from LKT Laboratories (St. Paul, MN, http://www.lktlabs.com), Nutlin-3 was from Calbiochem (Darmstadt, Germany, http://www.emdchemicals. com), and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were from Peprotech (Princeton, NJ, http://www.peprotech.com).

Techniques: Expressing, Western Blot, Transfection, Control, Staining, Small Interfering RNA

Journal: Stem cells (Dayton, Ohio)

Article Title: MEK-ERK signaling dictates DNA-repair gene MGMT expression and temozolomide resistance of stem-like glioblastoma cells via the MDM2-p53 axis.

doi: 10.1002/stem.753

Figure Lengend Snippet: Figure 2. MEK/ERK pathway inhibition reduces MGMT expression and temozolomide resistance of stem-like glioblastoma cells. (A): The stem-like glioblastoma cells treated with or without 10 lM SL327 for 3 days were subjected to immunoblot analysis for the expression of MGMT and phospho-ERK. (B): The stem-like glioblastoma cells were transfected with the combination of siRNAs against MEK1 and MEK2 or with a nontargeting control (Cont.). After 3 days of transfection, the cells were analyzed by immunoblotting with the indicated antibodies. (C): The stem-like glioblastoma cells pretreated with or without 10 lM SL327 for 3 days were subsequently treated with or without temozolomide (TMZ, 50 lM) for 4 hours and then subjected to cell death assay 72 hours after the temozolomide treatment. The upper panels show representa- tive images of the assay (red ¼ dead cells, blue ¼ live cells), and the graph shows the percentage of dead cells (means þ standard deviations of three independent experiments). *, p < .01, **, p < .05, n.s., not significant. Abbreviations: DMSO, dimethyl sulfoxide; ERK, extracellular sig- nal-regulated kinase; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; MGMT, O6-methylguanine DNA methyltrans- ferase; siRNA, short-interfering RNA; TMZ, temozolomide.

Article Snippet: SL327 was purchased from Enzo Life Sciences (New York, NY, http://www.enzolifesciences.com), temozolomide was from LKT Laboratories (St. Paul, MN, http://www.lktlabs.com), Nutlin-3 was from Calbiochem (Darmstadt, Germany, http://www.emdchemicals. com), and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were from Peprotech (Princeton, NJ, http://www.peprotech.com).

Techniques: Inhibition, Expressing, Western Blot, Transfection, Control, Small Interfering RNA

Journal: Stem cells (Dayton, Ohio)

Article Title: MEK-ERK signaling dictates DNA-repair gene MGMT expression and temozolomide resistance of stem-like glioblastoma cells via the MDM2-p53 axis.

doi: 10.1002/stem.753

Figure Lengend Snippet: Figure 5. Effective elimination of tumor-initiating population of stem-like glioblastoma cells by combination of MEK inhibitor and temozolomide treatments. TGS01 stem-like glioblastoma cells pre- treated with or without SL327 (10 lM) for 3 days were subsequently treated with or without TMZ (50 lM) for 4 hours and then, 72 hours after temozolomide treatment, implanted orthotopically into the brains of nude mice (five mice per group), as detailed in Materials and Meth- ods section. Survival of mice was evaluated by Kaplan-Meier analysis. Abbreviations: DMSO, dimethyl sulfoxide; MEK, mitogen-activated protein/extracellular signal-regulated kinase kinase; TMZ, temozolomide.

Article Snippet: SL327 was purchased from Enzo Life Sciences (New York, NY, http://www.enzolifesciences.com), temozolomide was from LKT Laboratories (St. Paul, MN, http://www.lktlabs.com), Nutlin-3 was from Calbiochem (Darmstadt, Germany, http://www.emdchemicals. com), and epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were from Peprotech (Princeton, NJ, http://www.peprotech.com).

Techniques:

Journal: Molecular cancer therapeutics

Article Title: Small Molecule BMH-compounds that Inhibit RNA Polymerase I and Cause Nucleolar Stress

doi: 10.1158/1535-7163.MCT-14-0256

Figure Lengend Snippet: BMH-9, BMH-22 and BMH-23 cause nucleolar segregation and RPA194 degradation. A, Chemical structures of BMH-9, BMH-22 and BMH-23. B and C, A375 melanoma cells were incubated for 3 hours with BMH-9, BMH-22 and BMH-23 (10 μM), and ActD (50 ng/ml), fixed and stained for B, UBF and FBL and C, NPM and NCL. Representative single cell images of N = 5 experiments are shown. D, Cells were incubated for 3 hours with BMH-9, BMH-22 and BMH-23 (10 μM), and ActD (50 ng/ml), or were pretreated with proteasome inhibitor MG132 (10 μM) for 30 minutes, fixed and stained for RPA194. Representative images of N = 3 experiments are shown. Scale bars, 10 μm. E, Cells were treated as in D, and additionally, with Nutlin-3 (10 μM). Cell lysates were analyzed by Western blotting for RPA194, TAFI110 and TIF-IA. Representative experiment of N = 4 is shown. F, A375 cells were treated with cycloheximide (CHX) in the presence or absence of the BMH-molecules (10 μM) for the indicated times, analyzed by western blotting and quantified. RPA194 signals were normalized to NCL used as a loading control.

Article Snippet: Actinomycin D was from Sigma, Nutlin-3 was obtained from Alexis Biochemicals and Sigma, and MG132 from Biomol International LP.

Techniques: Incubation, Staining, Western Blot

Journal: Molecular cancer therapeutics

Article Title: Small Molecule BMH-compounds that Inhibit RNA Polymerase I and Cause Nucleolar Stress

doi: 10.1158/1535-7163.MCT-14-0256

Figure Lengend Snippet: p53 is dispensable for nucleolar stress. A, SaOS-2 cells were treated with BMH-9, BMH-22 and BMH-23 (10 μM) and ActD (50 ng/ml) for 3 hours and stained for UBF, NCL and RPA194. Scale bars 10 μm. B, p53 isogenic HCT116 cells were treated with BMH-9, BMH-22, BMH-23 and Nutlin-3 (5 μM) for the indicated times and cells were counted. N = 2 experiments conducted in triplicate. Fold change to control are shown as mean ± SEM. C, SaOS-2 cells were treated with BMH-9, BMH-22, BMH-23 and Nutlin-3 (10 μM) or irradiated (IR) with 10 Gy and incubated for 72 hours. Cells were fixed and stained with propidium iodine and analyzed by flow cytometry. Cell cycle distribution was analyzed, and is shown in the inset. Separately, the fraction of sub-G1 cells was analyzed and is shown as percentage of cells in the total population. D, A375 cells were treated with BMH-9, BMH-22 and BMH-23 (0.5 μM and 5 μM) and in combination with Nutlin-3 (0.5 μM and 5 μM) and incubated for 24 hours. Viability was determined using the WST-1 assay. N = 2 experiments conducted in triplicate. Combination index (CI) was determined according to Chou-Talalay (35). Dashed line indicates additive effect, and values < 1 represent synergism. E, Schematic drawing of BMH-compound effects.

Article Snippet: Actinomycin D was from Sigma, Nutlin-3 was obtained from Alexis Biochemicals and Sigma, and MG132 from Biomol International LP.

Techniques: Staining, Irradiation, Incubation, Flow Cytometry, WST-1 Assay