human aml leukemia cell lines nb4  (DSMZ)

Journal: Cancer Gene Therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: A NB4 cells were treated with different doses of ATO for 24 h, and apoptosis was detected by flow cytometry. B PML-RARα in the NB4 cells was detected by immunoblot after treating with different doses of ATO for 12 h. C ROS level in NB4 induced by ATO for 4 h was detected by using 10 μM DCFH-DA. D Quantification of ROS level in NB4 induced by ATO for 4 h. Normalized to control (0 μM ATO). E NB4 cells were treated with 1.5 μM ATO, 10 or 20 μM H 2 O 2 for 4 h, and then ROS level was detected by using 10 μM DCFH-DA. F Quantification of ROS level in NB4 induced by 1.5 μM ATO, 10 or 20 μM H 2 O 2 for 4 h. Normalized to control. G Quantification of ROS level in NB4 induced by 1.5 μM ATO, 10 or 20 μM H 2 O 2 for 24 h. Normalized to control. H The apoptosis ratio of NB4 cells treated with 1.5 μM ATO or different doses of H 2 O 2 for 24 h was detected by flow cytometry. I NB4 cells were pre-treated with 8 mM NAC or 10 mM GSH for 1 h, and then 2 μM ATO was added for another 24 h. Apoptosis was detected by flow cytometry. Two-tailed Student t test, ** P < 0.01, *** P < 0.001, **** P < 0.0001, the ns indicate no significant difference. Error bars reflect ± SEM of three independent experiments.

Article Snippet: Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Flow Cytometry, Western Blot, Control, Two Tailed Test

Journal: Cancer Gene Therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: A Ubiquitinated proteins were detected by immunoblot after treatment with 1 or 1.5 μM ATO for 16 h. B , C ProteoStat kit was used to detect protein aggregates in NB4 cells by flow cytometry after treatment with 1 or 1.5 μM ATO ( B ) or 10 or 20 μM H 2 O 2 ( C ) for 16 h. Normalized to control. D Cell viability of indicated AML cell lines with ATO treatment after 24 h. E The global protein synthesis of indicated AML cell lines was detected by Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit. F Correlation between global protein synthesis and IC 50 . G The global protein synthesis of NB4 cells treated with 0.25 μg/mL cycloheximide, 25 μM rapamycin, 0.2 μg/mL puromycin, or 100 nM anisomycin for 20 h was detected by Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit. H Quantification of global protein synthesis (Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit) in NB4 treated with indicated protein synthesis inhibitors for 20 h. Normalized to control. I NB4 cells were pre-treated with 0.25 μg/mL cycloheximide, 25 μM rapamycin, 0.2 μg/mL puromycin, or 100 nM anisomycin for 1 h, and then 1.5 μM ATO was added for another 24 h. Apoptosis was detected by flow cytometry. Two-tailed Student t test, * P < 0.05, ** P < 0.01, *** P < 0.001, the ns indicate no significant difference. Error bars reflect ± SEM of three independent experiments.

Article Snippet: Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Western Blot, Flow Cytometry, Control, Two Tailed Test

Journal: Cancer Gene Therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: A The NB4 cells were pre-treated with 0.1 μg/mL puromycin for 1 h, and then 4 μM Biotin-As was added for another 16 h. Immunofluorescence visualized the localization of puromycin, Biotin-As, and aggresome in the cells. Scale bar, 25 μm. B The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then 20 μM puromycin was added for another 30 min. Cell fractions were prepared for the Co-IP assays using the streptavidin magnetic beads and indicated antibodies. C Immunoblot of indicated proteins in the NB4 cells at 0, 4, 8, 12, and 24 h with 0.5 or 2 μM ATO treatment. D The NB4 cells were pre-treated with 0.5 μM PERKi (GSK2656157) or 10 μM GCN2iB for 1 h, and then 2 μM ATO was added for another 4 h. Immunoblot of indicated proteins in the NB4 cells.

Article Snippet: Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Immunofluorescence, Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: Cancer Gene Therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα–JNK pathway

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: A Polysome profiles from NB4 cells with or without 1.5 μM ATO treatment for 6 h. B Immunoblot of indicated proteins in the NB4 cells at 0, 4, 20 h with 0.5 or 2 μM ATO treatment. C NB4 cells were pre-treated with 5 μM JNK inhibitor (JNK-IN-8) for 1 h, and then 2.5 μM ATO was added for another 24 h. Apoptosis was detected by flow cytometry. D Immunoblot of ZAKα and p-ZAKα in the NB4 cells at 4 or 20 h with 0.5 or 2 μM ATO treatment. The phosphorylated proteins in the cell lysate were enriched by Phos-tag TM Agarose. E The apoptosis ratio of ZAKα-knockdown NB4 cells with or without ATO treatment for 24 h. F Immunoblot of ZAKα in the ZAKα-knockdown NB4 cells. G The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then cell fractions were prepared for the Co-IP assays using the streptavidin magnetic beads and indicated antibodies. H The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then 20 μM puromycin was added for another 30 min. Cell fractions were prepared for the Co-IP assays using the NEMF, p97, and puromycin antibodies. I The NB4 cells were pre-treated with or without 1 μM NMS873 for 1 h and then indicated doses of ATO were added for another 24 h. The apoptosis ratio was detected by flow cytometry. Two-tailed Student t test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Error bars reflect ± SEM of three independent experiments.

Article Snippet: Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Western Blot, Flow Cytometry, Knockdown, Co-Immunoprecipitation Assay, Magnetic Beads, Two Tailed Test

Journal: Cancer gene therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα-JNK pathway.

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: Fig. 3 ATO targets nascent polypeptides while activating the GCN2–ATF4 pathway. A The NB4 cells were pre-treated with 0.1 μg/mL puromycin for 1 h, and then 4 μM Biotin-As was added for another 16 h. Immunofluorescence visualized the localization of puromycin, Biotin- As, and aggresome in the cells. Scale bar, 25 μm. B The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then 20 μM puromycin was added for another 30 min. Cell fractions were prepared for the Co-IP assays using the streptavidin magnetic beads and indicated antibodies. C Immunoblot of indicated proteins in the NB4 cells at 0, 4, 8, 12, and 24 h with 0.5 or 2 μM ATO treatment. D The NB4 cells were pre-treated with 0.5 μM PERKi (GSK2656157) or 10 μM GCN2iB for 1 h, and then 2 μM ATO was added for another 4 h. Immunoblot of indicated proteins in the NB4 cells.

Article Snippet: Cell lines and cell culture Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Co-Immunoprecipitation Assay, Magnetic Beads, Western Blot

Journal: Cancer gene therapy

Article Title: Arsenic trioxide and p97 inhibitor synergize against acute myeloid leukemia by targeting nascent polypeptides and activating the ZAKα-JNK pathway.

doi: 10.1038/s41417-024-00818-z

Figure Lengend Snippet: Fig. 4 ATO causes ribosome stalling and p97 inhibitor increases the sensitivity of APL to ATO. A Polysome profiles from NB4 cells with or without 1.5 μM ATO treatment for 6 h. B Immunoblot of indicated proteins in the NB4 cells at 0, 4, 20 h with 0.5 or 2 μM ATO treatment. C NB4 cells were pre-treated with 5 μM JNK inhibitor (JNK-IN-8) for 1 h, and then 2.5 μM ATO was added for another 24 h. Apoptosis was detected by flow cytometry. D Immunoblot of ZAKα and p-ZAKα in the NB4 cells at 4 or 20 h with 0.5 or 2 μM ATO treatment. The phosphorylated proteins in the cell lysate were enriched by Phos-tagTM Agarose. E The apoptosis ratio of ZAKα-knockdown NB4 cells with or without ATO treatment for 24 h. F Immunoblot of ZAKα in the ZAKα-knockdown NB4 cells. G The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then cell fractions were prepared for the Co-IP assays using the streptavidin magnetic beads and indicated antibodies. H The NB4 cells were treated with 1 μM Biotin-As for 4 h, and then 20 μM puromycin was added for another 30 min. Cell fractions were prepared for the Co-IP assays using the NEMF, p97, and puromycin antibodies. I The NB4 cells were pre-treated with or without 1 μM NMS873 for 1 h and then indicated doses of ATO were added for another 24 h. The apoptosis ratio was detected by flow cytometry. Two-tailed Student t test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars reflect ± SEM of three independent experiments.

Article Snippet: Cell lines and cell culture Human AML leukemia cell lines NB4, MV4-11, NOMO-1, MOLM-13, THP-1, U937, HL-60, and Kasumi-1 were purchased from DSMZ.

Techniques: Western Blot, Cytometry, Knockdown, Co-Immunoprecipitation Assay, Magnetic Beads, Two Tailed Test

Journal: JCI Insight

Article Title: Biallelic TET2 mutations confer sensitivity to 5 ′ -azacitidine in acute myeloid leukemia

doi: 10.1172/jci.insight.150368

Figure Lengend Snippet: ( A ) Parental TET2 monoallelic HEL cell clones (HEL TET2 monoallelic; unfilled symbols) and TET2 CRISPR/Cas9-mutated HEL cell clones (HEL TET2 biallelic; filled symbols) were cultured in soft agar supplemented with 1 μM 5′-Aza (left), 20 nM Ara-C (center), or 20 nM daunorubicin (right), and CE (relative to respective vehicle control–treated cells) was determined after 30 days. Mean and SD of indicated number of clones from 3 independent experiments shown. P values calculated by Student’s t test (2-tailed). ( B ) Parental TET2 monoallelic HEL cell clones (HEL TET2 monoallelic; unfilled symbols) and TET2 CRISPR/Cas9-mutated HEL cell clones (HEL TET2 biallelic; filled symbols) were treated with 5′-Aza (left), Ara-C (center), or daunorubicin (right), and cell density (relative to respective vehicle control–treated cells) was determined after 96 hours. Data represent mean and SD of indicated number of clones from 3 independent experiments. P values calculated by 2-way ANOVA. ( C ) Western blot showing cleaved PARP in HEL cells with monoallelic and biallelic TET2 mutations following exposure to 2 mM 5′-Aza over 48 hours. GAPDH was used as a loading control. ( D ) Western blot (top) showing TET2 protein expression in a panel of 10 AML cell lines. GAPDH was used as a loading control. TET2 protein expression was quantified in each cell line and plotted against 5′-Aza IC 50 (left) and IC 90 (right) values. AU were measured by the Fuji LAS-300 Image Analyzer.

Article Snippet: HEL, THP-1, HL-60, AML2, Kasumi, MV4-11, AML3, U937, SKM1, and NB4 AML cell lines were obtained from DSMZ.

Techniques: Clone Assay, CRISPR, Cell Culture, Control, Western Blot, Expressing

Journal: JCI Insight

Article Title: Biallelic TET2 mutations confer sensitivity to 5 ′ -azacitidine in acute myeloid leukemia

doi: 10.1172/jci.insight.150368

Figure Lengend Snippet: ( A ) Mean synergy scores for verapamil and 5′-Aza combination and for ( B ) tariquidar and 5′-Aza in combination, stratified by TET2 mutant allele dosage. Synergy scores for verapamil/5′-Aza and tariquidar/5′-Aza were significantly higher for TET2 monoallelic mutant HEL cell clones compared with TET2 biallelic HEL cell clones ( P = 0.0003 and P < 0.0001, respectively, paired 2-tailed Student’s t test). Data are derived from 6 independent experimental replicates using 2 independent cell clones for each TET2 genotype. Data represent the mean and SD of indicated number of clones. ( C ) HEL AML cell clones were exposed to escalating doses of 5′-Aza to generate significantly resistant subclones ( P = 0.0008, unpaired 2-tailed Student’s t test). Data show IC 50 values for 5′-Aza–resistant subclones derived from parental cells with either TET2 monoallelic mutation (unfilled symbols) or TET2 biallelic mutation (filled symbols). Data represent the mean and SD of indicated number of clones. ( D ) Western blots show ABCB1 protein levels in 3 representative 5′-Aza–resistant derivatives from cells with either TET2 monoallelic mutation (left) or TET2 biallelic mutation (right). ( E ) ABCB1 protein levels were quantified in parental HEL cells, and 9 independent 5′-Aza–resistant derivatives with either TET2 monoallelic mutation (unfilled symbols) or TET2 biallelic mutation (filled symbols). ABCB1 protein levels were quantified and normalized to GAPDH, with the expression in each parental cell given a nominal value of 1. ABCB1 protein levels of 5′-Aza–resistant derivatives were significantly higher than their respective parental cells ( P = 0.0069, paired 2-tailed Student’s t test). Solid horizontal line represents the median fold change in ABCB1 protein expression in 5′-Aza–resistant subclones relative to their respective parental cells (represented by the dashed horizontal line).

Article Snippet: HEL, THP-1, HL-60, AML2, Kasumi, MV4-11, AML3, U937, SKM1, and NB4 AML cell lines were obtained from DSMZ.

Techniques: Mutagenesis, Clone Assay, Derivative Assay, Western Blot, Expressing