apl derived nb4 cell line  (DSMZ)

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Western blot analysis of basal PML-RARα and USP22 expression in wild-type (wt), control (non-human target; n.h.t) and USP22 knockout (KO) NB4 acute promyelocytic leukemia (APL) cells. β-Actin served as loading control. Representative blots of at least two different independent experiments are shown. B Densitometric quantification of gray level intensities of the major (130 kDa) PML-RARα isoform detected by Western blot analysis in wt, n.h.t and USP22 KO NB4 APL cells, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. C Basal mRNA expression levels of the PML-RARα long isoform in wt, n.h.t. and USP22 KO NB4 APL cells using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to n.h.t. Mean and SEM of three independent experiments are shown. D Western blot analysis of wt, n.h.t and USP22 KO NB4 APL cells treated with 20 μg/ml CHX for the indicated timepoints. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. E Densitometric quantification of gray level intensities of the major (130 kDa) PML-RARα isoform detected by Western blot analysis in wt, n.h.t and USP22 KO NB4 APL cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. F Western blot analysis of n.h.t and USP22 KO HEK293T cells, transiently transfected with plasmids encoding the long isoform of PML-RARα and treated with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. G Densitometric quantification of gray level intensities of the long isoform of PML-RARα transiently transfected in n.h.t and USP22 KO HEK293T cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown. H Western blot analysis of HEK293T cells, transiently transfected with plasmids encoding the long isoform of wt (PML-RARα) and K394R (KR) PML-RARα (PML-K394R-RARα) and treated with 20 μg/ml CHX for the indicated timepoints. Co-transfection with GFP plasmid served as transfection control. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. Red dashed line indicates protein stability at 50% compared to untreated controls. I Densitometric quantification of gray level intensities of the long isoform of wt (PML-RARα) and K394R (KR) PML-RARα (PML-K394R-RARα) transiently transfected in HEK293T cells treated with 20 μg/ml CHX for the indicated timepoints, normalized against loading control intensities. Mean and SEM of three independent experiments are shown.

Article Snippet: Human colon carcinoma HT-29, human embryonic kidney HEK293T and human APL NB4 cell lines were obtained from and authenticated by DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Expressing, Control, Knock-Out, Quantitative RT-PCR, Gene Expression, Transfection, Cotransfection, Plasmid Preparation

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Western blot analysis of RARα and USP22 in wild-type (wt), control (non-human target; n.h.t) and USP22 knockout (KO) NB4 APL cells treated with the indicated concentrations of all- trans retinoic acid (ATRA) for 120 h. β-actin served as loading control. Representative blots of at least two different independent experiments are shown. B Basal and ATRA-induced mRNA expression levels of PML-RARα in wt, n.h.t and USP22 KO NB4 APL cells incubated with the indicated ATRA concentrations for 120 h using qRT-PCR. UT, untreated. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to UT of wt NB4 cells Mean and SEM of three independent biological replicates are shown. C FACS analysis of wt, n.h.t., and USP22 KO NB4 APL cells incubated with the indicated ATRA concentrations for 120 h. Shown is the cell count per fluorescence intensity of PE-labeled CD11b. D Mean fluorescence intensities (MFIs) of CD11b-PE signals on wt, n.h.t., and USP22 KO NB4 APL cells incubated with the indicated ATRA concentrations for 120 h. UT, untreated. Mean and SEM of four independent biological replicates are shown. E Basal and ATRA-induced mRNA expression levels of CD11b in wt, n.h.t and USP22 KO NB4 APL cells incubated with the indicated ATRA concentrations for for 120 h using qRT-PCR. UT, untreated. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to UT of wt NB4 cells Mean and SEM of three independent biological replicates are shown.

Article Snippet: Human colon carcinoma HT-29, human embryonic kidney HEK293T and human APL NB4 cell lines were obtained from and authenticated by DSMZ (Braunschweig, Germany).

Techniques: Western Blot, Control, Knock-Out, Expressing, Incubation, Quantitative RT-PCR, Gene Expression, Cell Counting, Fluorescence, Labeling

Journal: Cell Death Discovery

Article Title: USP22 regulates APL differentiation via PML-RARα stabilization and IFN repression

doi: 10.1038/s41420-024-01894-8

Figure Lengend Snippet: A Basal and ATRA-induced mRNA expression levels of IRF1 in wild-type (wt), control (non-human target; n.h.t) and USP22 knockout (KO) NB4 APL cells incubated with the indicated ATRA concentrations for 120 h using qRT-PCR. UT, untreated. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to UT of wt NB4 cells. Mean and SEM of three independent biological replicates are shown. B Basal mRNA expression levels of USP22 and the indicated ISGs in n.h.t and USP22 KO NB4 APL cells using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to n.h.t. Mean and SEM of three independent biological replicates are shown. C Model of USP22-mediated effects on PML-RARα and IFN signaling in APL.

Article Snippet: Human colon carcinoma HT-29, human embryonic kidney HEK293T and human APL NB4 cell lines were obtained from and authenticated by DSMZ (Braunschweig, Germany).

Techniques: Expressing, Control, Knock-Out, Incubation, Quantitative RT-PCR, Gene Expression

Journal: Biochemical pharmacology

Article Title: The clinically relevant CHK1 inhibitor MK-8776 induces the degradation of the oncogenic protein PML-RARα and overcomes ATRA resistance in acute promyelocytic leukemia cells.

doi: 10.1016/j.bcp.2023.115675

Figure Lengend Snippet: Fig. 1. Effects of CHK1 inhibition by MK-8776 in APL cells. Sensitivity of NB4, R4, R2, and THP1 cells to the MK-8776 inhibitor. Each cell line was treated with 0–32 µM MK-8776 for 24, 48, 72, and 96 h. Cells were counterstained with propidium iodide (PI) and acquired for volumetric absolute counting. Approximately 10.000 useful events were acquired for each experimental point, and the absolute number of live cells (A) and death cells (B) were analyzed. (A) Graphs represent the percentage of cells surviving fractions normalized to untreated cells for each concentration of MK-8776. For each cell line and for each time point the IC50 values were calculated. (B) Graphs represent the percentage of PI-positive cells, which corresponds to death cells. (C) CD11b expression in NB4, R4, and R2 cells treated with MK- 8776. NB4 cells were treated with 1.6 µM MK-8776 for 72 h, R4 cells were treated with 4 µM MK-8776 for 96 h, and R2 cells were treated with 4 µM MK-8776 for 48 h. Cells were hybridized with the anti-CD11b antibody labeled with phycoerythrin (PE). In orange, the population of cells expressing the CD11b surface antigen. All the cell lines were also tested also for their responsiveness to 1 μM ATRA for 96 h. (D) NBT assay performed in R4 cells treated with 4 μM MK-8776 for 96 or with the only vehicle as control. Absorbance was measured at 570 nm. Mean values were derived from repeated experiments ± SD (Student’s t-test, **p ≤0.01). (E) Morphological changes induced in R4 cells treated with 4 μM MK-8776 for 96 h, as revealed by the May-Grünwald/Giemsa staining (scale bar: 10 μm). (F) R4 and R2 cells were treated with 4 μM MK-8776 for 48, 72, and 96 h. Protein lysates were analyzed by immunoblot using the anti-RARα antibody, which allows the detection of PML-RARα protein. As positive control to evaluate PML-RARα degradation, NB4 cells were treated with 1 μM ATRA for 96 h. (G) Synergy map of R4 cells treated for 96 h with MK-8776 (0, 0.5, 1, 2, and 4 μM) and ATO (0, 0.01, 0.1, and 1 μM). The effect of the treatments was measured by the NBT assay and the synergy index was calculated with the Combenefit freeware software [31]. (H) The graph reports the synergic effect of the most effective combination of R4 cells treatment with 4 μM MK-8776 and 0.01 μM ATO. Mean values were derived from repeated experiments ± SD (Student’s t-test, *p ≤0.05, ***p ≤0.001, and ****p ≤0.0001).

Article Snippet: The human APL-derived NB4 cell line bears the t(15;17) translocation and expresses the fusion protein PML-RARα [46] (DSMZ, Braunschweig, Germany).

Techniques: Inhibition, Concentration Assay, Expressing, Labeling, Control, Derivative Assay, Staining, Western Blot, Positive Control, Software

Journal: bioRxiv

Article Title: A novel retinoic acid analog, 4-Amino-2-Trifluoromethyl-Phenyl Retinate (ATPR) triggers differentiation and is effective in the treatment of Acute Promyelocytic Leukemia

doi: 10.1101/2020.07.09.194928

Figure Lengend Snippet: A. Chemical structure of ATPR, a novel all-trans retinoic acid (ATRA) derivative, designed and synthesized by our team. B. Concentration-dependent inhibition of cellular viability of ATRA (1*10 -6 mol/L), ATPR (1*10 -9 , 1*10 -8 , 1*10 -7 , 1*10 -6 , 1*10 -5 mol/L) on NB4 cells as measured by CCK-8 assay. * p <0.05, ** p <0.01 versus control group. C. NB4 cells were treated with ATPR (1*10 -7 , 1*10 -6 , 1*10 -5 mol/L) and ATRA (1*10 -6 mol/L). The cell cycle distribution was analyzed by flow cytometry. * p <0.05, ** p <0.01 versus control group.

Article Snippet: The APL cell line NB4 was purchased from Genechem Co.Ltd. (Shanghai, China).

Techniques: Synthesized, Concentration Assay, Inhibition, CCK-8 Assay, Control, Flow Cytometry

Journal: bioRxiv

Article Title: A novel retinoic acid analog, 4-Amino-2-Trifluoromethyl-Phenyl Retinate (ATPR) triggers differentiation and is effective in the treatment of Acute Promyelocytic Leukemia

doi: 10.1101/2020.07.09.194928

Figure Lengend Snippet: NB4 cells were treated with ATPR (1*10 -7 , 1*10 -6 , 1*10 -5 mol/L) and ATRA (1*10 -6 mol/L). CD11b expression was analyzed by flow cytometry. * p <0.05, ** p <0.01 versus control group.

Article Snippet: The APL cell line NB4 was purchased from Genechem Co.Ltd. (Shanghai, China).

Techniques: Expressing, Flow Cytometry, Control