human myeloid nb4  (CLS Cell Lines Service GmbH)

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Activation Assay, Staining, Western Blot, Inhibition

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Membrane, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Disruption, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Article Snippet: Cell culture and CD44 ligation Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence

Journal: Cell reports

Article Title: ATP7B-maintained copper stores in myeloid progenitors are required for functional maturation of neutrophils

doi: 10.1016/j.celrep.2026.116955

Figure Lengend Snippet: (A) Co-immunostaining of ATP7B (green) with the trans-Golgi marker TGN46 (red) in mouse myelocytes and co-immunostaining of ATP7B (green) with the cis-Golgi marker GM130 (red) in human myeloid NB4 cells. A zoomed view used to demonstrate the ATP7B (green) vesicular pattern. (B) Co-immunostaining of ATP7B (yellow) with TGN46 (cyan), the early endosome marker EEA1 (cyan), the lysosome marker LAMP1 (magenta), and the late endosomal marker Rab8 (magenta) in non-differentiated NB4 cells. There was no significant co-localization of ATP7B with Golgi and endosomal markers, respectively. Scale bar, 5 μm. (C) Live images of CopperGREEN sensor in non-differentiated NB4 cells. DAPI (blue) was used as a nuclear marker. Scale bar, 10 μm. See also – .

Article Snippet: Human myeloid NB4 (Cytion, cat# 300299) and NB4/Luc+ cells (AML-M3, gift from Gabriel Ghiaur’s lab) were cultured in RPMI1640 media supplemented with 10% FBS +2 mM Glutamine +1% Pen-Strep.

Techniques: Immunostaining, Marker

Journal: Cell reports

Article Title: ATP7B-maintained copper stores in myeloid progenitors are required for functional maturation of neutrophils

doi: 10.1016/j.celrep.2026.116955

Figure Lengend Snippet: (A) Schematic representation of ATRA-induced human myeloid NB4 cell differentiation. (B) Western blot analysis reveals that ATP7B protein abundance is significantly reduced and CEBPε is upregulated during ATRA-induced NB4 cell differentiation from day 0 to day 6. β-actin was used as an endogenous loading control. Immunoblotting data represent 4–5 independent experiments. (C) Densitometric analysis demonstrating altered protein abundance of ATP7B and CEBPε was performed using ImageJ by normalizing to β-actin. (D) Western blot analysis reveals that ATP7A protein abundance is upregulated during ATRA-induced NB4 cell differentiation from day 0 to day 6. (E) Densitometric analysis demonstrating altered protein abundance of ATP7A was performed using ImageJ by normalizing to β-actin. (F–L) mRNA expression of CEBPε (F), ATP7B (G), ATP7A (H), CTR1 (I), CTR2 (J), LOXL2 (K), and AOC2 (L) at day 0, day 2, and day 6 of ATRA-induced NB4 cell differentiation. Values represent mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the undifferentiated control at day 0 or between multiple time points analyzed by ANOVA. Data represent 3–5 independent experiments. The gray color bar denotes day 0, magenta denotes day 2, and blue denotes day 6 NB4 cells, respectively. Abbreviations: ATRA, all-trans retinoic acid. See also .

Article Snippet: Human myeloid NB4 (Cytion, cat# 300299) and NB4/Luc+ cells (AML-M3, gift from Gabriel Ghiaur’s lab) were cultured in RPMI1640 media supplemented with 10% FBS +2 mM Glutamine +1% Pen-Strep.

Techniques: Cell Differentiation, Western Blot, Quantitative Proteomics, Control, Expressing

Journal: Cell reports

Article Title: ATP7B-maintained copper stores in myeloid progenitors are required for functional maturation of neutrophils

doi: 10.1016/j.celrep.2026.116955

Figure Lengend Snippet: (A) Live images of CopperGREEN in ATRA-induced differentiated NB4 cells (day 6). Scale bar, 10 μm. (B) Cellular copper content at day 0, day 2, and day 6 of ATRA-induced NB4 cell differentiation, measured by ICP-MS, and statistical analysis was performed by ordinary one-way ANOVA. Fg denotes femtogram. (C) CopperGREEN staining in isolated BM progenitors (CD117+) and BM neutrophils reveals an increase in intracellular Cu in BM neutrophils. Representative images of one of the CopperGREEN-stained images is shown from progenitors (CD117+) and neutrophils derived from BM. Scale bar, 10 μm. (D) Fluorescence intensity shows increased Cu levels in BM neutrophils compared to progenitors (CD117+). The data represent two independent experiments. **** p < 0.0001 by t test. (E) CopperGREEN staining was lower in blood neutrophils than in BM neutrophils. Treatment with the Cu chelator TTM (10 μM) reduced intracellular Cu staining in BM neutrophils. BM neutrophils without CopperGREEN were used as a negative control. The data represent three independent experiments. Scale bar, 10 μm.

Article Snippet: Human myeloid NB4 (Cytion, cat# 300299) and NB4/Luc+ cells (AML-M3, gift from Gabriel Ghiaur’s lab) were cultured in RPMI1640 media supplemented with 10% FBS +2 mM Glutamine +1% Pen-Strep.

Techniques: Cell Differentiation, Staining, Isolation, Derivative Assay, Fluorescence, Negative Control

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 treatment induces apoptosis in NB4 cells through activation of caspase-8. (A) Apoptosis induction. NB4 cells were treated with A3D8 at the indicated concentrations for 1 to 3 days. The percentage of apoptotic cells were determined by FACS after staining with annexin-V. The data shown are the mean plus SE of three independent experiments. (B) The levels of cleaved PARP, caspase-3, -8 and -9. NB4 cells were treated with 2.5 μg/ml A3D8 for 1 to 3 days and the relative levels of the indicated proteins were analyzed by Western blotting using specific antibodies. GAPDH levels were used as loading controls. (C) Inhibition of A3D8-induced apoptosis by caspase inhibitors. NB4 cells were pretreated with the pancaspase inhibitor Z-VAD (50 μM), the caspase-9 inhibitor Z-LETD (50 μM), the caspase-8 inhibitor Z-IETD (50 μM) for 4 h and then with 2.5 μg/ml A3D8 for 72 h. The percentage of apoptotic cells were detected by FACS after staining with annexin V. The data shown are the mean plus SE of three independent experiments.

Article Snippet: Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Activation Assay, Staining, Western Blot, Inhibition

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Fas is clustered into membrane lipid rafts in NB4 cells after A3D8 treatment. NB4 cells were treated with or without 2.5 μg/ml A3D8 or mouse IgG for 72 h. The cells were fixed and stained with the FITC-Ctx B subunit to identify lipid rafts (green fluorescence) and with an anti-Fas antibody to identify Fas (red fluorescence). Areas of colocalization between membrane rafts and Fas are yellow.

Article Snippet: Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Membrane, Staining, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: Disruption of lipid rafts with MCD abrogates A3D8-induced apoptosis in NB4 cells. NB4 cells were treated with A3D8 at 2.5 μg/ml for 2 days and then with 2.5 mg/ml MCD for 30 min. MCD was washed out and cells were treated with or without A3D8 2.5 μg/ml for another 24 h. Lipid rafts were determined with a confocal microscopy (A). Cells were fixed and stained with the FITC-Ctx B subunit to identify rafts (green fluorescence) and nuclei were identified by staining with DAPI. The percentage of apoptotic cells in NB cells treated with A3D8 and/or MCD was measured by FACS after staining with Annexin-V (B). The relative levels of cleaved caspase-3, -8 and PARP in NB cells treated with A3D8 and/or MCD were analyzed by Western blotting (C).

Article Snippet: Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Disruption, Confocal Microscopy, Staining, Fluorescence, Western Blot

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: HMWHA and J173 neither induce apoptosis nor induce clustering of lipid rafts in NB4 cells. NB4 cells were treated with HMWHA 350 μg/ml, J173 2.5 μg/ml, A3D8 2.5 μg/ml and dialyzed A3D8 (A3D8-D) 2.5 μg/ml for 72 h. The percentage of apoptotic cells was determined by FACS after staining with annexin-V (A). Lipid raft clustering was determined by confocal microscopy after staining with the FITC-CtxB subunit to identify lipid rafts (green fluorescence) and to identify nuclei by staining with DAPI (B).

Article Snippet: Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Staining, Confocal Microscopy, Fluorescence

Journal: Cancer Biology & Therapy

Article Title: CD44 ligation with A3D8 antibody induces apoptosis in acute myeloid leukemia cells through binding to CD44s and clustering lipid rafts

doi: 10.4161/cbt.21784

Figure Lengend Snippet: A3D8 and J173 antibodies have different binding abilities to HL-60, SKNO-1 and NB4 cells. (A) Western blot analysis of CD44 protein levels. Cellular lysates were isolated from HL-60, SKNO-1 and NB4 cells, subjected to 8% SDS-gel electrophoresis and then probed with either A3D8 or J173 antibody. (B) Cell surface CD44 binding of A3D8 and J173. HL-60, SKNO-1 and NB4 cells were incubated with mouse IgG, A3D8 and J173 first and then FITC labeled secondary antibody. The fluorescence strength was determined by FACS.

Article Snippet: Human myeloid leukemia cell lines NB4 (provided by Dr. M. Lanotte), 34 HL-60 (obtained from ATCC, VA) and SKNO-1 (provided by Dr. Y. Honma) 35 were cultured in RPMI 1640 medium supplemented with 100 units/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L L -glutamine and 10% (v/v) heat-inactivated fetal bovine serum (FBS).

Techniques: Binding Assay, Western Blot, Isolation, SDS-Gel, Electrophoresis, Incubation, Labeling, Fluorescence