Journal: Cancer Research

Article Title: NF-κB–Inhibited Acute Myeloid Leukemia Cells Are Rescued from Apoptosis by Heme Oxygenase-1 Induction

doi: 10.1158/0008-5472.can-09-3407

Figure Lengend Snippet: Figure 1. NF-κB subunits p50 and p65 are highly expressed in AML cells and are not killed by NF-κB inhibition. A, cytosolic and nuclear extracts were obtained from human AML samples and control cells, extracts were separated by SDS-PAGE, and Western blot analysis was conducted for p50, p52, p65, and c-Rel protein levels. Cytosolic blots were reprobed with GAPDH, and nuclear blots were reprobed with TATA-binding protein (TBP) to confirm equal loading. Nuclear extracts from different AML samples as well as control cells were assessed for p50 and p65 DNA binding using κB Transfactor ELISA (inset). B, AML and control cells were treated with increasing doses of BAY 11-7082 for 24 h. Cell number was assessed by MTS assay, and values indicate means ± SEM (n = 3). C, THP-1 cells were treated for various times with BAY 11-7082 (10 μmol/L) and nuclear extracts were prepared. Analysis by Western of p50 and p65 expression in these samples. D, where indicated, AML and control cells were treated with increasing doses of BAY 11-7082 alone or in combination with TNF for 24 h. Where indicated (*), statistical significance of P < 0.05 exists between the different treatment groups.

Article Snippet: NF-κB DNA binding was measured using the NF-κB p65 transcription factor ELISA kit (Panomics).

Techniques: Inhibition, Control, SDS Page, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, MTS Assay, Expressing

Journal: Cancer Research

Article Title: NF-κB–Inhibited Acute Myeloid Leukemia Cells Are Rescued from Apoptosis by Heme Oxygenase-1 Induction

doi: 10.1158/0008-5472.can-09-3407

Figure Lengend Snippet: Figure 2. Inhibiting NF-κB in AML cells induces HO-1 expression. A, human AML and control cells were treated with 10 μmol/L BAY 11-7082 for various times. RNA was extracted and HO-1 mRNA was measured using real-time PCR, with expression normalized to GAPDH mRNA levels. B, Western blot analysis was performed on nonmalignant primary monocytes, AML103, and THP-1 in response to BAY 11-7082 incubation to confirm RNA results. C, RNA was extracted from human AML cells and control cells; HO-1 mRNA was measured and normalized to GAPDH levels. *, statistical significance of P < 0.05 exists between the different treatment groups. D, THP-1 cells were transfected with 30 nmol/L of either control, p50, or p65 siRNA and incubated for 24 and 48 h. Extract p50, p65, and HO-1 protein levels were analyzed by Western blot. Blots were reprobed with β-actin to confirm equal loading.

Article Snippet: NF-κB DNA binding was measured using the NF-κB p65 transcription factor ELISA kit (Panomics).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Western Blot, Incubation, Transfection

Journal: Cancer Research

Article Title: NF-κB–Inhibited Acute Myeloid Leukemia Cells Are Rescued from Apoptosis by Heme Oxygenase-1 Induction

doi: 10.1158/0008-5472.can-09-3407

Figure Lengend Snippet: Figure 3. HO-1 transcription is negatively controlled by NF-κB in AML cells. A, schematic presentation of the HO-1 promoter constructs created for this study. B, THP-1 cells were transiently transfected with 1 μg of each promoter construct. Cells were cotransfected with pRL-CMV for normalization of transfection efficiency. Extracts were harvested, and then luciferase activity was measured. *, P < 0.01, deleted κB1 against control; **, P < 0.01, sulforaphane treated in the ARE-deleted construct against wild-type ARE. As a negative control, cells were cotransfected with HO-1 siRNA. As a positive control, sulforaphane (10 μmol/L) was used to activate the ARE. C, scheme of primer locations used to amplify genomic regions. ChIP analysis of the HO-1 promoter using antibodies against p50, p65, and Nrf2. Normal rabbit IgG was used as a control. THP-1 cells were untreated or treated with BAY 11-7082 for 8 h. D, real-time PCR was performed in triplicate on immunoprecipitated DNA and input DNA. Data presented as percent of input. Values are means ± SD (n = 4).

Article Snippet: NF-κB DNA binding was measured using the NF-κB p65 transcription factor ELISA kit (Panomics).

Techniques: Construct, Transfection, Luciferase, Activity Assay, Control, Negative Control, Positive Control, Real-time Polymerase Chain Reaction, Immunoprecipitation