Journal:

Article Title: Human Immunodeficiency Virus Type-1 Protein Tat Induces Tumor Necrosis Factor-?-Mediated Neurotoxicity

doi: 10.1016/j.nbd.2007.03.004

Figure Lengend Snippet: Tat-induced TNF-α release in differentiated monocytes was time and concentration-dependent. Tat1-72 (● 14 ng/ml; ■ 70 ng/ml; ◆ 140 ng/ml; ▴ 700 ng/ml) induced a rapid concentration-dependent increase in TNF-α from differentiated U937 (A) and THP-1 cells (C). Tat1-72 (open squares) and TatΔ31-61 (open circles) concentration-dependently increased levels of TNF-α from differentiated U937 (B) and THP-1 cells (D). Basal levels of TNF-α for U937 cells were 88.1 ± 8.2 pg/ml and for THP-1 cells 20.3 ± 5.6 pg/ml. Values are means and SEM. Tat1-72 time-course (n=3) and concentration-response (n=3) experiments, and TatΔ31-61 concentration-response (n=5) experiments for each cell line.

Article Snippet: Human pro-monocytic U937 (ATCC # CRL-1593.2) and human monocytic leukemia THP-1 cells (ATCC # TIB-202) were maintained in RPMI-1640 media supplemented with 10% heat-inactivated FBS and antibiotic/antimycotic at 37°C and 5% CO 2 .

Techniques: Concentration Assay

Journal:

Article Title: Human Immunodeficiency Virus Type-1 Protein Tat Induces Tumor Necrosis Factor-?-Mediated Neurotoxicity

doi: 10.1016/j.nbd.2007.03.004

Figure Lengend Snippet: Specificity of Tat1-72- and TatΔ31-61-induced increases in levels of TNF-α. Tat1-72- (1.4 μg/ml) and TatΔ31-61- (1.4 μg/ml) induced increases in levels of TNF-α from U937 cells were blocked significantly by immunoadsorption with anti-Tat antibody (Ab) and by heating the peptide at 100°C (100°C). For all conditions tested, values are means and SEM (n=4). * p < 0.05 versus Tat1-72 (A) or TatΔ31-61 (B).

Article Snippet: Human pro-monocytic U937 (ATCC # CRL-1593.2) and human monocytic leukemia THP-1 cells (ATCC # TIB-202) were maintained in RPMI-1640 media supplemented with 10% heat-inactivated FBS and antibiotic/antimycotic at 37°C and 5% CO 2 .

Techniques:

Journal:

Article Title: Human Immunodeficiency Virus Type-1 Protein Tat Induces Tumor Necrosis Factor-?-Mediated Neurotoxicity

doi: 10.1016/j.nbd.2007.03.004

Figure Lengend Snippet: Effects of supernatants of Tat1-72 and TatΔ31-61 treated U937 cells on neuronal viability. (A) Phase contrast with overlying fluorescence images of primary hippocampal neurons stained with calcein (green), ethidium homodimer (red) and Hoechst 33342 (blue). 24 h after media from U937 cells was added to neurons, viable neurons (arrows) exhibited green fluorescence staining after esterase hydrolysis of calcein-AM. Non-viable cells (arrow heads) exhibited chromatin condensation when stained with Hoechst 33342, were unable to exclude ethidium homodimer, and lacked neuritic processes. Bar, 50 μm. (B) Media from U937 cells (sup.) and to a significantly greater extent media from U937 cells treated with 1.4 μg/ml of Tat1-72 (Tat sup.) or TatΔ31-61 (mTat sup.) decreased significantly neuronal viability. Neuronal survival was expressed as a percent of control conditions. Values are means and SEM of determinations made using six separate batches of cultured cells (n=6).

Article Snippet: Human pro-monocytic U937 (ATCC # CRL-1593.2) and human monocytic leukemia THP-1 cells (ATCC # TIB-202) were maintained in RPMI-1640 media supplemented with 10% heat-inactivated FBS and antibiotic/antimycotic at 37°C and 5% CO 2 .

Techniques: Fluorescence, Staining, Control, Cell Culture

Journal:

Article Title: Human Immunodeficiency Virus Type-1 Protein Tat Induces Tumor Necrosis Factor-?-Mediated Neurotoxicity

doi: 10.1016/j.nbd.2007.03.004

Figure Lengend Snippet: TNF-α immunoneutralization blocked the neurotoxic effects of supernatants from Tat1-72 and TatΔ31-61 treated U937 cells. Immunoneutralization of TNF-α with anti-TNF-α antibody (1 μg/ml) decreased significantly neuronal cell death caused by supernatants from U937 cells treated with 1.4 μg/ml Tat1-72 (Tat sup. vs. Tat sup. + Ab) and supernatants from U937 cells treated with 1.4 μg/ml TatΔ31-61 (mTat sup. vs. mTat sup. + Ab). Neuronal survival was expressed as a percent of values obtained for neurons treated with media from vehicle-treated U937 cells (sup). Values are means and SEM of determinations made using four separate batches of cultured cells (n=4). Bar, 50 μm.* p < 0.05 sup vs. Tat sup. or mTat sup.

Article Snippet: Human pro-monocytic U937 (ATCC # CRL-1593.2) and human monocytic leukemia THP-1 cells (ATCC # TIB-202) were maintained in RPMI-1640 media supplemented with 10% heat-inactivated FBS and antibiotic/antimycotic at 37°C and 5% CO 2 .

Techniques: Cell Culture

Journal: Protein engineering, design & selection : PEDS

Article Title: The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines.

doi: 10.1093/protein/gzs011

Figure Lengend Snippet: Fig. 4. Rv3166c peptide binding activity profile. Amino acid sequence and specific U937 and A549 cell binding activity for Rv3166c synthesised peptides. Peptides are shown on the left-hand side, numbers indicating their position in the native protein; Tyr was added at the C-terminal end of those peptides lacking it. The black bars on the right-hand side represent each peptide’s binding activity determined as the specific binding/total peptide added ratio. The dotted line separates peptides having 1% binding activity (HABPs).

Article Snippet: The A549 (ATCC CLL-185) and U937 (ATCC CRL-2367) cell lines were kept in Roswell Park Memorial Institute (RPMI) 1640 medium culture supplemented with 10% fetal bovine serum (FBS) at 378C and 5% CO2.

Techniques: Binding Assay, Activity Assay, Sequencing

Journal: Protein engineering, design & selection : PEDS

Article Title: The role of Mycobacterium tuberculosis Rv3166c protein-derived high-activity binding peptides in inhibiting invasion of human cell lines.

doi: 10.1093/protein/gzs011

Figure Lengend Snippet: Fig. 5. A549 and U937 cell invasion inhibition assays. HABP 16 295, 16 296 and 16 298 inhibition of A549 cell invasion percentages are shown at three concentrations (5, 50 and 100 mM). HABPs 16 287 and 16 296 U937 cell invasion inhibition percentages are shown at the same three concentrations. About 10 mM cytochalasin was used as positive invasion inhibition control in both cases.

Article Snippet: The A549 (ATCC CLL-185) and U937 (ATCC CRL-2367) cell lines were kept in Roswell Park Memorial Institute (RPMI) 1640 medium culture supplemented with 10% fetal bovine serum (FBS) at 378C and 5% CO2.

Techniques: Inhibition, Control

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 1 (a) U937 cells were incubated for 24 h in the presence of the indicated concentration of flavopiridol (FP; nm) or MG-132 (MG; nm), after which the percentage of cells exhibiting apoptotic morphology was determined by evaluating Wright–Giemsa-stained cytospin preparations. (b) U937 cells were incubated for 24 h in the presence of 100 nm FP 7300 nm MG, 500 nm lactacystin (LC), or 5 mm LLnL, respectively, after which the percentage of cells exhibiting apoptotic morphology was determined as above. (c) U937 cells were treated as above for 24 h, after which the percentage of cells exhibiting reduced mitochondrial membrane potential (DCm) was determined by monitoring DiOC6 uptake. (d) U937 cells were treated with 100 nm FP 7300 nm MG for 24 h, after which cells were washed free of drug and plated in soft agar as described in Materials and methods. After 12 days incubation, colonies were scored, and colony formation for each condition expressed relative to untreated control cells. Results represent the means7s.d. for three separate experiments performed in triplicate

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Incubation, Concentration Assay, Staining, Membrane, Control

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 2 (a, b) Leukemic blasts were obtained from the peripheral blood of two patients with acute myelogenous leukemia (FAB classification M2; 485% blasts in each case), incubated in medium þ 10% FBS in the presence of the indicated concentrations of flavopiridol7MG-132 for 24 h, and apoptosis assessed by evaluating Wright–Giemsa-stained cytospin preparations. (c) U937 cells were incubated for 24 h in the presence of 12 mm roscovitine (Ros)75 nm bortezomib (btzmb), after which the percentage of apoptotic cells was determined by Annexin V-FITC staining and flow cytometry. Results represent the means7s.d. for three separate experiments performed in triplicate. (d) U937 cells were exposed to a range of concentrations of roscovitine (8–14 mm) and bortezomib (4–7 nm) alone and in combination at fixed ratio (e.g., 2000 : 1) for 24 h. At the end of this period, the percentage of cells exhibiting apoptotic morphology was determined for each condi- tion; fractional effect (FA) values were determined by comparing results to those of untreated controls, and median dose–effect analysis employed to characterize the nature of the interaction. CI values less than 1.0 denote a synergistic interaction. Two additional studies yielded equivalent results

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Incubation, Staining, Cytometry

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 3 U937 cells were treated for 24 h with 100 nm flavopir- idol7300 nm MG-132 (a), or 7500 nm lactacystin (b), after which cells were lysed and subjected to Western blot using the indicated antibodies. CF ¼ cleavage fragment. Alternatively, cytosolic frac- tions were obtained, and expression of cytochrome c and Smac/ DIABLO were monitored by Western blot. Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to tubulin or actin to ensure equal loading and transfer. Two additional studies yielded equivalent results

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Western Blot, Expressing

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 5 U937 cells were treated with 100 nm flavopiridol7300 nm MG-132 for 24 h, after which expression of phosphorylated MAP kinases (e.g. SAPK/JNK, ERK, and p38) (a), cell cycle regulators (e.g. p34cdc2, p21CIP/WAF1, p27KIP1), and apoptotic regulators (e.g. Bcl- 2, Bcl-xL, Bax, XIAP, and Mcl-1) (b) were monitored by Western blot. Alternatively, cell lysates were subjected to SAPK/JNK kinase assay. SAPK/JNK activity was reflected by phosphorylated GST-c-Jun (a, bottom panel). Similarly, U937 cells were treated with 100 nm flavopiridol7500 nm lactacystin for 24 h (c), after which expression of indicated protein was analysed by Western blot. Phosphorylation of SAPK/JNK was also monitored in HL- 60, Jurkat, and Raji cells after treatment with 150 nm flavopir- idol þ 500 nm MG-132 for 24 h (d). Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to tubulin or actin to ensure equal loading and transfer. Results are representative of three separate experiments

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Expressing, Western Blot, Kinase Assay, Activity Assay, Phospho-proteomics

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 6 Upper panels: U937 cells were incubated for 24 h with 100 nm flavopiridol þ 300 nm MG-132 or þ 500 nm lactacystin in the presence of either the SAPK/JNK peptide inhibitor D-JNKI1 (1 mm) or its control peptide D-TAT (1 mm), after which the percentage of cells exhibiting apoptotic morphology and reduced DCm was evaluated (a); alternatively, SAPK/JNK phosphorylation and PARP cleavage were monitored by Western blot (b). CF ¼ cleavage fragment. Middle panels: U937 cells were transfected with JNK1 siRNA oligonucleotides or controls, treated with 100 nm flavopiridol þ 300 nm MG-132 for 24 h, after which the percentage of cells exhibiting apoptotic morphology was determined. Values represent the means7s.d. for three separate experiments. In addition, cells were lysed and subjected to Western blot to monitor expression of JNK 1 and 2 and phospho-JNK, as described above (c). Alternatively, the percentage of apoptotic cells was monitored by Annexin V-FITC staining and flow cytometry; values represent the percentage of cells in the right lower and upper quadrants (annexin V þ and annexin V/PI þ) (d). Lower panels: U937 cells were treated with 100 nm flavopiridol7the SAPK/JNK activator anisomycin (30 ng/ml) for 24 h, after which the percentage of cells exhibiting apoptotic morphology and reduced DCm (e), as well as SAPK/JNK phosphorylation and PARP cleavage (f) were determined as above. For (a, c, e), values represent the means7s.d. for three separate experiments performed in triplicate. *Significantly lower than values for cells treated with D-TAT (a; Po0.01) or for cells without JNK1 siRNA (c; Po0.02), or greater than values for cells treated with flavopiridol alone (e; Po0.01). For (b, d and f), two additional experiments yielded equivalent results. For all Western blots, each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to actin to ensure equal loading and transfer

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Incubation, Control, Phospho-proteomics, Western Blot, Transfection, Expressing, Staining, Cytometry

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 7 U937 cells were stably transfected with full-length Bcl-2 cDNA or empty vector (pCEP); ectopic overexpression of Bcl-2 protein in U937/Bcl-2 cells is shown by Western blot (a, inset). Cells were treated with 25 mm VP-16 (6 h), or 100 nm flavopir- idol þ 300 nm MG-132 or þ 500 nm lactacystin (24 h), after which the percentage of cells exhibiting reduced DCm (a) or apoptotic morphology (b) was determined. The values represent the means7s.d. for three separate experiments performed in triplicate. *Significantly lower than values for pCEP cells; Po0.02 (a) and Po0.01 (b). **Not significant less than values for pCEP cells; P40.05 (a, b). Alternatively, after treatment with flavopiri- dol þ MG-132 as above, cells were lysed and subjected to Western blot to monitor phosphorylation of SAPK/JNK and cleavage of PARP (c). CF ¼ cleavage fragment. Two additional studies yielded equivalent results

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Over Expression, Western Blot, Phospho-proteomics

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 8 U937 cells stably transfected with DN-caspase 8, CrmA, or empty vector (pcDNA 3.1) were treated with 5 ng/ml TNFa. 1 mm cycloheximide (CHX) (4 h), or 100 nm flavopiridol þ 300 nm MG-132 or þ 500 nm lactacystin (24 h), after which the percentage of cells exhibiting reduced DCm (a) or apoptotic morphology (b) was determined. Values represent the means7s.d. for three separate experiments performed in triplicate. *Significantly lower than values for 3.1 cells; Po0.02 (a, b). Alternatively, U937/ caspase 8 (DN) and /pcDNA 3.1 cells were treated with flavopiridol þ MG-132 or þ lactacystin as above, after which cells were lysed and subjected to Western blot to monitor phosphoryla- tion of SAPK/JNK and cleavage of caspase-8 (c). Two additional studies yielded equivalent results

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Western Blot

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 9 (a) U937 cells were left untreated or treated with 300 nm MG-132 (upper left panel) for 24 h or 10 ng/ml TNFa for 15 h (lower panel) in the presence or absence of 100 nm flavopiridol, after which nuclear extracts were prepared and subjected to EMSA, as described in Materials and methods. Supershift analysis was performed using specific anti-p50 and anti-p65 antibodies to determine the identity of NF-kB-containing complexes (upper right panel). For C þ C0, 100-fold excess of unlabeled NF-kB oligonucleotides was coincubated for 10 min with the nuclear extract obtained from untreated cells prior to addition of labeled NF-kB oligonucleotides. Two additional studies yielded equivalent results. (b) U937 cells were stably transfected with IkBa mutant (Ser32, 36/Ala) or an empty vector (pcDNA3.1). Two U937 cell clones stably expressing IkBa mutant (2H6, 2C8), as well as empty vector controls (pCDNA 3.1) were treated with 5 ng/ml TNFa for 5 min and then subjected to EMSA as above. (c) U937/IkBa mutant and/pcDNA 3.1 cells were incubated for 24 h in the present of 50–125 nm flavopiridol, after which the percentage of cells exhibiting apoptotic morphology was determined. Values represent the means7s.d. for three separate experiments performed in triplicate. (d) U937/IkBa mutant (clone 2H6) and /pcDNA 3.1 cells were treated with 50 nm flavopiridol for 24 h, after which cells were lysed and subjected to Western blot; alternatively, cytosolic fractions were prepared to monitor release of mitochondrial cytochrome c and Smac/DIABLO. CF ¼ cleavage fragment. Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to actin to ensure equal loading and transfer. Results are representative of three separate experiments

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Labeling, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation, Clone Assay, Expressing, Incubation, Western Blot

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 10 (a) U937 cells were treated for 24 h with 75 nm flavopiridol73 mm Bay 11-7082 or 7250 nm PDTC, after which the percentage of cells exhibiting apoptotic morphology was determined. (b) U937 cells were treated for 24 h with 75 nm flavopiridol in the presence of either NF-kB-inhibitory peptide (SN50, 150 mg/ml) or its inactive control (SN50M, 150 mg/ml), after which the percentage of apoptotic cells was determined as above. (c) U937 cells were treated with flavopiridol7Bay 11-7082 as above, after which whole-cell lysates were subjected to Western blot. In addition, cytosolic fractions were prepared to monitor release of mitochondrial cytochrome c and smac/DIABLO. Two additional studies yielded equivalent results. Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to actin to ensure equal loading and transfer. For (a, b), the values represent the means7s.d. for three separate experiments performed in triplicate

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Control, Western Blot

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

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.

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: Reverse Transcription Polymerase Chain Reaction

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.

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: Activation Assay, Incubation, Western Blot, Expressing

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.

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: Activation Assay, Incubation, Staining

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.

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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Anti-inflammatory Role of Carotenoids in Endothelial Cells Derived from Umbilical Cord of Women Affected by Gestational Diabetes Mellitus

doi: 10.1155/2019/8184656

Figure Lengend Snippet: Effect of carotenoids on TNF- α -induced monocyte interaction in C- and GD-HUVECs. Monocyte-HUVEC adhesion in C- and GD-HUVECs untreated (Basal) and incubated for 24 h with BC or Lyc (2.5 μ mol/L) and then stimulated for 16 h with or without TNF- α (1 ng/mL). In the histogram (upper side), quantitative data express the number of U937 cells adhering within a high-power field (3.5mm 2 ). Each measurement is expressed as the mean ± SD of adhering cells from 3 experiments ( n = 3), each consisting of 8 counts per condition. In the lower side, representative photos of C- and GD-HUVECs for each experimental condition. ANOVA and Bonferroni multiple comparison test: ∗ p < 0.05 vs. basal C-HUVECs, ∗∗ p < 0.05 vs. TNF- α C-HUVECs, ∗∗∗ p < 0.05 vs. Basal GD-HUVECs, # p < 0.05 vs. TNF- α GD-HUVECs. Student'st-test: † p < 0.0002 basal GD-HUVECs vs. basal C-HUVECs, ‡ p < 0.0001 TNF- α GD-HUVECs vs. TNF- α C-HUVECs.

Article Snippet: The cells were grown to confluence in six-well tissue culture plates and U937 cell lines (European Collection of Authenticated Cell Cultures (ECACC)) were used to evaluate the adhesion to HUVEC monolayers as previously described [ ].

Techniques: Incubation, Comparison