u937 (DSMZ)

DSMZ u937
$Fig. 1. UNBS1450 induces apoptotic cell death in <t>U937</t> cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantiﬁcation (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.$
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$Fig. 1. UNBS1450 induces apoptotic cell death in U937 cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantiﬁcation (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.$

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 1. UNBS1450 induces apoptotic cell death in U937 cells. (A) Molecular structure. (B) Analysis of UNBS1450-induced cell death was performed by Trypan Blue staining after 24, 48 and 72 h of treatment with UNBS1450 at 10, 15, 20 and 30 nM. (C) Cell cycle analysis after 24 h of incubation time with indicated concentrations. (D) Hoechst staining (upper panel) and quantiﬁcation (lower panel) of the fraction of cells presenting fragmented nuclei. (E) Flow cytometry analysis after 24 h of incubation time at indicated concentrations. (F) Analysis of PBMCs viability after UNBS1450 treatment. PBMCs were seeded at 2 106, then after 24 h treated for 24 h with various concentrations (0–100 nM) of UNBS1450. Cells were then stained either by Trypan Blue (upper panel) or by Hoechst (panel below) to analyze either cell integrity or apoptosis induction. The data shown here were representative for three independent experiments.

Article Snippet: K562 (human chronic myelogenous leukemia), U937 (histiocytic lymphoma), Jurkat (T-cell leukemia), Raji (Burkitt’s Lymphoma), Hel (erythroleukemia), Molt (human acute lymphoblastic leukemia), Meg01 (human megacaryoblastic cells), HL60 (human promyelo- cytic leukemia), TF1 (erythroleukemia) and KBM5 (chronic myelogenous leukemia) cells (DSMZ) were cultured in RPMI medium (Lonza, Verviers, Belgium) supplemented with 10% (v/v) fetal calf serum (Lonza, Verviers, Belgium) and 1% (v/v) antibiotic–antimycotic (BioWhittaker, Verviers, Belgium) at 37 8C and 5% of CO2.

Techniques: Staining, Cell Cycle Assay, Incubation, Flow Cytometry

Fig. 2. Na+/K+-ATPase subunit a1 mRNA quantiﬁcation. Na+/K+-ATPase subunit a1 mRNA content of untreated PBMCs and a wide panel of hematological cancer cell lines including K562, Jurkat and U937 cells was transcribed and then quantiﬁed by RT-PCR. The quantiﬁcation of three independent experiments is expressed in brute 2^DCt values S.D.

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 2. Na+/K+-ATPase subunit a1 mRNA quantiﬁcation. Na+/K+-ATPase subunit a1 mRNA content of untreated PBMCs and a wide panel of hematological cancer cell lines including K562, Jurkat and U937 cells was transcribed and then quantiﬁed by RT-PCR. The quantiﬁcation of three independent experiments is expressed in brute 2^DCt values S.D.

Techniques: Reverse Transcription Polymerase Chain Reaction

Fig. 3. (A) Caspase activation. U937 cells were incubated in RPMI + 10% FCS UNBS1450 20 nM up to 24 h. Western blot analysis of UNBS1450-induced cleavage of pro-caspases-9, -8, -7 and -3. (B) Analysis of expression levels of anti- apoptotic proteins. UNBS1450-induced expression level alterations of XIAP, Bcl-2 and Mcl-1. The data shown here were representative for three independent experiments.

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 3. (A) Caspase activation. U937 cells were incubated in RPMI + 10% FCS UNBS1450 20 nM up to 24 h. Western blot analysis of UNBS1450-induced cleavage of pro-caspases-9, -8, -7 and -3. (B) Analysis of expression levels of anti- apoptotic proteins. UNBS1450-induced expression level alterations of XIAP, Bcl-2 and Mcl-1. The data shown here were representative for three independent experiments.

Techniques: Activation Assay, Incubation, Western Blot, Expressing

Fig. 4. UNBS1450 enables Bak/Bax activation. U937 cells were incubated for 24 h in RPMI + 10% FCS in presence or in absence of UNBS1450. Bak (A.) and Bax (B.) activation status were assessed by using primary antibodies speciﬁcally targeting activated forms of Bak (Ab-1; Calbiochem) and Bax (6A7; Santa Cruz). Counterstaining was done by Hoechst staining to assess apoptotic nuclei. The data shown here were representative for three independent experiments with similar results.

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 4. UNBS1450 enables Bak/Bax activation. U937 cells were incubated for 24 h in RPMI + 10% FCS in presence or in absence of UNBS1450. Bak (A.) and Bax (B.) activation status were assessed by using primary antibodies speciﬁcally targeting activated forms of Bak (Ab-1; Calbiochem) and Bax (6A7; Santa Cruz). Counterstaining was done by Hoechst staining to assess apoptotic nuclei. The data shown here were representative for three independent experiments with similar results.

Techniques: Activation Assay, Incubation, Staining

$Fig. 5. Inhibition by UNBS1450 of TNFa-induced NF-kB activation. (A) K562 and (B) Jurkat cells were pretreated with UNBS1450 at various concentrations from 10 to 50 nM and incubated for 2 h, followed by TNFa addition (20 ng/ml) and an additional incubation period of 6 h. Results are represented as the ratio of the measured luminescence of the ﬁreﬂy luciferase vector divided by the measured luminescence of the Renilla plasmid. Untreated cells were used as a negative control, cells treated with TNFa only as a positive control. Results are presented as mean S.D. of 3 individual measurements performed in triplicates. (C) Effect of UNBS1450 on the binding afﬁnity of NF-kB was assessed by an EMSA on the K562 and Jurkat cell lines. The data shown here were representative for three independent experiments with similar results. (D) For supershift/immunodepletion experiments, the nuclear extracts and labelled probes were incubated in the reaction mixture for 30 min on ice prior to a 30 min incubation with 2 mg of anti-p50 or anti-p65 antibodies. SS designates supershifted bands. (E) Jurkat cells were incubated with UNBS1450 (40 nM) for 2 h, followed by a TNFa (20 ng/ml) activation for the indicated time periods. Cytoplasmic and nuclear extracts were prepared, fractionated on a 10% SDS-page gel, transferred to a membrane and then tested for IkBa and p65. Protein loading and purity of nuclear/cytosolic extracts were veriﬁed by lamin B and a-tubulin Western blots. Data shown are representative for three independent experiments with similar results. K562 (F), and U937 (G) cells were incubated for 2 h in RPMI + 10% FCS in presence or in absence of various concentrations (10–50 nM) of UNBS1450 before being activated by TNFa during 22 h. After 24 h of incubation IL-8 concentrations in supernatants were measured. Untreated cells served as negative control whereas cells activated by TNFa only were used as a positive control. The data shown here were representative for three independent experiments with similar results.$

Journal: Biochemical pharmacology

Article Title: UNBS1450, a steroid cardiac glycoside inducing apoptotic cell death in human leukemia cells.

doi: 10.1016/j.bcp.2010.08.025

Figure Lengend Snippet: Fig. 5. Inhibition by UNBS1450 of TNFa-induced NF-kB activation. (A) K562 and (B) Jurkat cells were pretreated with UNBS1450 at various concentrations from 10 to 50 nM and incubated for 2 h, followed by TNFa addition (20 ng/ml) and an additional incubation period of 6 h. Results are represented as the ratio of the measured luminescence of the ﬁreﬂy luciferase vector divided by the measured luminescence of the Renilla plasmid. Untreated cells were used as a negative control, cells treated with TNFa only as a positive control. Results are presented as mean S.D. of 3 individual measurements performed in triplicates. (C) Effect of UNBS1450 on the binding afﬁnity of NF-kB was assessed by an EMSA on the K562 and Jurkat cell lines. The data shown here were representative for three independent experiments with similar results. (D) For supershift/immunodepletion experiments, the nuclear extracts and labelled probes were incubated in the reaction mixture for 30 min on ice prior to a 30 min incubation with 2 mg of anti-p50 or anti-p65 antibodies. SS designates supershifted bands. (E) Jurkat cells were incubated with UNBS1450 (40 nM) for 2 h, followed by a TNFa (20 ng/ml) activation for the indicated time periods. Cytoplasmic and nuclear extracts were prepared, fractionated on a 10% SDS-page gel, transferred to a membrane and then tested for IkBa and p65. Protein loading and purity of nuclear/cytosolic extracts were veriﬁed by lamin B and a-tubulin Western blots. Data shown are representative for three independent experiments with similar results. K562 (F), and U937 (G) cells were incubated for 2 h in RPMI + 10% FCS in presence or in absence of various concentrations (10–50 nM) of UNBS1450 before being activated by TNFa during 22 h. After 24 h of incubation IL-8 concentrations in supernatants were measured. Untreated cells served as negative control whereas cells activated by TNFa only were used as a positive control. The data shown here were representative for three independent experiments with similar results.

Techniques: Inhibition, Activation Assay, Incubation, Luciferase, Plasmid Preparation, Negative Control, Positive Control, Binding Assay, Immunodepletion, SDS Page, Membrane, Western Blot

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