Journal: Molecular oncology
Article Title: KMT2A degradation is observed in decitabine-responsive acute lymphoblastic leukemia cells.
doi: 10.1002/1878-0261.13792
Figure Lengend Snippet: Fig. 4. Influence of Decitabine (DEC) on biological processes in acute B-lymphoblastic leukemia cell lines SEM, RS4;11, REH, and NALM-6. (A) To assess proliferation, cells were incubated with 100 nM DEC or control (DMSO) for 72 h before trypan blue staining. SEM, REH: n = 3; RS4;11, NALM-6: n = 4, mean SD; paired t test of absolute cell counts. (B) WST-1 assay for the analysis of cellular metabolism following 72 h incubation with 100 nM DEC. SEM, REH: n = 3; RS4;11, NALM-6: n = 4, mean SD; paired t test of absolute signal intensities. (C) Analysis of apoptotic processes after 72 h of 100 nM DEC using Annexin V/propidium iodide (PI) staining, and flow cytometry. n = 3, mean SD; paired t test. (D) Cell cycle phases (G2/M, S, G0/G1) were determined by PI staining, and subsequent flow cytometry after 72 h DEC incubation (100 nM). n = 3, mean SD; paired t test. (E, G) Probe-based gene expression analysis of CDKN2A (E), and CDKN1B (G) following 72 h 1 μM DEC incubation. n = 3, mean SD; paired t test of ΔCt vs. GAPDH values. (F, H) Quantitative analysis of p16 (F), and p27 (H) protein expression after 72 h incubation with 1 μM DEC. n = 3, mean SD; ratio paired t test of absolute GAPDH-normalized signal intensities. *P < 0.05; **P < 0.01.
Article Snippet: Human B—ALL cell lines SEM (RRID:CVCL_0095), RS4;11 (RRID:CVCL_0093), REH (RRID: CVCL_1650), and NALM-6 (RRID:CVCL_0092) as well as human AML lines MV4;11 (RRID: CVCL_0064), and MOLM13 (RRID:CVCL_2119) were purchased from DSMZ (Braunschweig, Germany), and maintained at 37 °C and 5% CO2 in IMDM medium (SEM), Alpha MEM medium (RS4;11), or RPMI medium (REH, NALM-6, MV4;11, MOLM13) supplemented with 10% heat-inactivated fetal calf serum, and 100 μg mL 1 penicillin/streptomycin (all PAN—biotech, Aidenbach, Germany).
Techniques: Incubation, Control, Staining, WST-1 Assay, Cytometry, Gene Expression, Expressing
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![Fig. 1. Decitabine (DEC)-induced effects on methyltransferase activity, gene and protein expression. (A) 3D crystal protein structure of DNMT1 (gray) in complex with DNA (red). The DNA-binding CXXC domain is colored in green. RCSB Protein data bank PDB identifier 3PTA [6,74]. (B) Crystal structure of the KMT2A CXXC domain (green) bound to human DNA (red). RCSB Protein data bank PDB identifier 2KKF [75,76]. (C) Amino acid (aa) sequences of the DNMT1 (top), and KMT2A (bottom) CXXC domains were retrieved from uniprot [77]. Shared amino acids were analyzed using BLASTP suite-2sequences [78] search, and depicted in the middle box. (D–G) Acute B-lymphoblastic leukemia cell lines <t>SEM,</t> <t>RS4;11,</t> REH, and NALM-6 were incubated with 1 μM DEC or vehicle substance (DMSO) for 72 h. (D) Influence on global methylation was analyzed by methylation-specific qPCR of the LINE-1 retrotransposon. n = 3, mean SD; paired t test. (E) Quantitative analysis of histone 3, lysine residue 4 trimethylation (H3K4me3) by western blot was used to determine the global transcription rate. n = 3, mean SD; ratio paired t test of absolute GAPDH-normalized signal intensities. (F) Probe-based gene expression analysis of DNMT1, and KMT2A. Both, the N-terminal CXXC-domain as well as the C-terminal SET domain have been analyzed. n = 3, mean SD; paired t test of ΔCt vs. GAPDH values. (G) Quantitative analysis of DNMT1, and KMT2A protein expression. n = 3, mean SD; ratio paired t test of absolute GAPDH-normalized signal intensities. *P < 0.05; **P < 0.01.](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4404/pm39754404/pm39754404__page5_image1.jpg)
