Journal: Molecular and Clinical Oncology

Article Title: Analysis of genes encoding epigenetic regulators in myeloproliferative neoplasms: Coexistence of a novel SETBP1 mutation in a patient with a p.V617F JAK2 positive myelofibrosis

doi: 10.3892/mco.2019.1840

Figure Lengend Snippet: Human cancer cell lines tested.

Article Snippet: Informed consent was obtained from all patients and procedures were approved by the Ethical Committee on Clinical Research of University of Navarra. table ft1 table-wrap mode="anchored" t5 Table II. caption a7 Cell line name Repository (number) Origin A-549 DSMZ (ACC-107) Lung carcinoma BK-006 ECACC p.V617F JAK2 positive PV BK-013 ECACC (98100924) p.V617F JAK2 negative ET BK015 ECACC (99092421) p.V617F JAK2 negative ET DAUDI DSMZ (ACC-78) Burkitt lymphoma EOL-1 DSMZ (ACC-386) Acute myeloid eosinophilic leukemia F-36P DSMZ (ACC-543) Acute myeloid leukemia secondary to myelodysplastic syndrome (MDS) HCC-1937 DSMZ (ACC-513) Breast carcinoma HEL DSMZ ACC-11 Erythroleukemia HL60 DSMZ ACC-3 Acute myeloid leukemia (AML) HU3 Dr Morgan, USA Acute megakaryoblastic leukemia K-562 DSMZ ACC-10 BCR-ABL1 positive chronic myeloid leukemia (CML) in blast crisis KARPAS-299 DSMZ ACC-31 T cell lymphoma KARPAS-422 DSMZ ACC-32 B cell lymphoma M-07e DSMZ ACC-104 Acute megakaryoblastic leukemia MG-63 ATCC: CRL-1427 Osteosarcoma MOLM-13 DSMZ ACC-554 Acute myeloid leukemia (AML) MOLT-16 DSMZ ACC-29 T cell acute lymphoid leukemia (T-ALL) MOLT-4 DSMZ ACC-362 Acute lymphoid leukemia (ALL) MV-411 DSMZ ACC-102 Acute monoblastic/monocytic leukemia RAJI DSMZ ACC-319 Burkitt lymphoma REH DSMZ ACC-22 B-ALL (t(12;21) (p13;q22) (fusion ETV6-RUNX1 )) SET2 DSMZ ACC-608 p.V617F JAK2 positive ET TF-1 DSMZ ACC-334 Erythroleukemia UKE-1 Dr W. Fiedelr, Hamburg, Germany p.V617F JAK2 positive ET transformed to AML Open in a separate window ECACC, European Collection of Cell Culture; DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen.

Techniques: Transformation Assay

Journal: Nature Communications

Article Title: Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML

doi: 10.1038/s41467-024-48953-3

Figure Lengend Snippet: A EVI1 and ∆EVI1 expression in EVI1 High AML cell lines ( n = 3 biological replicates). B Nuclear localization expression of EVI1 (in red) detected by immunofluorescence (IF) in EVI1 High AML cell lines ( n = 3 biological replicates). Cell nuclei were stained with DAPI (blue), scale bar: 10 µm. C EVI1 and ∆EVI1 expression in TF1 cells 6 days after shRNA transduction. D Percentage of EVI1 mRNA relative to the control gene RPL13A (ΔΔCT) in TF1 cells 6 days after shRNA transduction. E Effect of EVI1 loss in TF1 cells at three or six days after shRNA selection on cell proliferation. F Effect of shRNA-directed EVI1 loss in the ability of TF1 to form colonies in methylcellulose compared to NT ( n = 2 biological replicates). Scale bar: 1000 µm. The histogram on the right shows the mean number of colonies per field 20 days after plating cells. NT = non-targeting, sh#16 and sh#87 = shRNA directed against EVI1 ( C – F ). G Effect of AR-42, belinostat, and entinostat on cell viability in EVI1 High cell lines following 72 hours (hr) of treatment. H Annexin V/propidium iodide (PI) staining after 72 hr of HDACis treatment. Events ≥ 20,000. I Apoptotic fold increase, expressed as a percentage of annexin V-positive cells relative to vehicle control. J Effect of HDACis on the ability of 3q26 AML cell lines to form colonies in methylcellulose. Representative images of UCSD/AML1 treated with vehicle (DMSO), 0.5 µM AR-42, 0.5 µM belinostat, and 0.5 µM entinostat ( n = 3 biological replicates). Scale bar: 1000 µm. K The histogram shows the mean number of colonies per field 20 days after plating cells ( n = 3 biological replicates). Statistical significance among groups was determined by one-way ( D , K ) or two-way ( E ) ANOVA using Tukey’s correction for multiple comparison testing. Data are presented as mean ± standard deviation (SD) in D ( n = 3), E ( n = 4), F ( n = 10 fields per condition), G ( n = 2), and K ( n = 10 fields per condition). Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: The human cell lines MOLM1 (#ACC 720), UCSD/AML1 (#ACC 691), HNT34 (#ACC 600), TF1 (#ACC 334), MUTZ-3 (#ACC 295), 293 T (#ACC 635), OCI/AML3 (#ACC 582), MOLM13 (#ACC 554), NOMO1 (#ACC 542), OCI/AML2 (#ACC 99), GDM1 (#ACC 87), HL-60 (#ACC 3), SKM1 (#ACC 547) and 5637 (#ACC 35) were purchased from the Leibniz-Institut DSMZ-German collection of microorganisms and cell cultures (Germany).

Techniques: Expressing, Immunofluorescence, Staining, shRNA, Transduction, Control, Selection, Comparison, Standard Deviation

Journal: Nature Communications

Article Title: Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML

doi: 10.1038/s41467-024-48953-3

Figure Lengend Snippet: A EVI1, ∆EVI1, and cleaved caspase 3 expression in 3q26 EVI1 High cell lines after 24 hr of treatment with DMSO or HDACis ( n = 2 biological replicates). B Representative IF images of EVI1 (red) in 3q26 EVI1 High cell lines (left, n = 3 biological replicates). Cell nuclei were stained with DAPI (blue), scale bar: 100 µm. Right: fluorescence intensity of nuclear EVI1 content in 3q26 EVI1 High AML cell lines after 24 hr of treatment. C IF images of U937T_E10 cells cultured in the presence (EVI1 Low , top panel) or absence (EVI1 High , bottom panel) of tetracycline, incubated with an anti-EVI1 (red) antibody ( n = 2 biological replicates). Nuclei in blue (DAPI). Scale bar: 100 µm. D Effect of HDACis and ara-C on cell viability after 72 hr of treatment in U937T_E10 cell cultured in the presence (EVI1 Low , black line) or absence (EVI1 High , red line) of tetracycline. E Representative IF showing HL-60 cells transduced with an empty (top panels) or an ORF- EVI1 cDNA (bottom panels) vector and stained with an anti-EVI1 antibody (in red, n = 2 biological replicates). Nuclei in blue (DAPI). Scale bar: 100 μm. F Effect of HDACis and ara-C on cell viability after 72 h of treatment in HL-60 ± ORF- EVI1 cDNA. G Abnormal 3q26 pattern on fluorescence in situ hybridization (FISH) in MOLM1 sorted by cell size. The break-apart hybridization pattern 1F1G1O (one fusion and two separated signals, one green and one orange) indicates the break and split of the EVI1 locus. The abnormal pattern was observed in cells with large nuclei. At least 100 nuclei/cells were analyzed ( n = 2 biological replicates). Scale bar: 20 μm. H Expression of EVI1 in MOLM1 sorted based on cell size. Large cells express EVI1 compared to small cells. I Effects of HDACis and chemotherapy treatment on viability in MOLM1 EVI1 High and MOLM1 EVI Low after 72 h of drug exposure. J Effect of AR-42, belinostat, entinostat, ara-C, vincristine, methotrexate, and daunorubicin on EVI1 High (MOLM1, UCSD/AML1, HNT34, TF1, and MUTZ-3) and EVI1 Low (NOMO1, MOLM13, OCI/AML1, OCI/AML2, GDM1, SKM1, and IMS-M2) AML cell lines calculated using the area under the curve (AUC) model of the log-transformed dose-response data. A lower AUC corresponds to greater sensitivity. Statistical significance was determined by two-tailed non-parametric t-test (Mann-Whitney) (J ), one-way ANOVA with Tukey’s correction for multiple comparison testing ( B ). Data are presented as mean ± SD in B (UCSD/AML1 n = 828, MOLM1 n = 1028, HNT34 n = 571), D ( n = 2), F ( n = 2), I ( n = 2) and J (EVI1 High n = 5, EVI1 Low n = 7). Source data are provided as a Source Data file. See also Supplementary Fig. .

Article Snippet: The human cell lines MOLM1 (#ACC 720), UCSD/AML1 (#ACC 691), HNT34 (#ACC 600), TF1 (#ACC 334), MUTZ-3 (#ACC 295), 293 T (#ACC 635), OCI/AML3 (#ACC 582), MOLM13 (#ACC 554), NOMO1 (#ACC 542), OCI/AML2 (#ACC 99), GDM1 (#ACC 87), HL-60 (#ACC 3), SKM1 (#ACC 547) and 5637 (#ACC 35) were purchased from the Leibniz-Institut DSMZ-German collection of microorganisms and cell cultures (Germany).

Techniques: Expressing, Staining, Fluorescence, Cell Culture, Incubation, Transduction, Plasmid Preparation, In Situ Hybridization, Hybridization, Transformation Assay, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Communications

Article Title: Orthogonal proteogenomic analysis identifies the druggable PA2G4-MYC axis in 3q26 AML

doi: 10.1038/s41467-024-48953-3

Figure Lengend Snippet: A Circle plot showing chromatin-associated proteins immunoprecipitated with an anti-EVI1 antibody by RIME. The bar length indicates the mean of mass spectrometry spectral count (SPEC) of uniquely identified proteins of two biological replicates per condition. Bar colors indicate protein ontology of the EVI1 interactors. B Histograms show the normalized gene expression levels of SERBP1 , RPL14 , RPL18 , RPS2 , PA2G4 , RPS10 , FBL , PABPC1 , RACK1 , RSL1D1 , and DDX21 following 16 hr of 0.5 µM AR-42, 2 µM entinostat in UCSD/AML1, or 0.8 µM AR-42 and 4 µM entinostat in MOLM1 compared to vehicle. Statistical significance was determined using DESeq2. Whiskers show median values (central black lines) and 25 th and 75 th percentiles (bottom and top bounds), respectively. The bars represent values that exceed 1.5 times the interquartile range (IQR) from the edge of each box: (vehicle n = 6, treated n = 12). C Tracks showing EVI1 binding and H3K27ac enrichment across the DDX21, FBL, and PA2G4 locus in HNT34 cells treated with vehicle (DMSO), AR-42 and entinostat. The bottom bar represents the genes (hg19), and the y-axis represents normalized read density scaled to 1 million. D PA2G4, EVI1, ∆EVI1, and MYC expression in TF1, HNT34, and UCSD/AML1 cells 6 days after shRNA transduction ( n = 3 biological replicates). E Effect of PA2G4 overexpression on HNT34 cells treated with HDACis. Western blot analysis showing PA2G4, EVI1, ∆EVI1, and MYC in wild-type or PA2G4 -overexpressing HNT34 cells after 24 h of treatment with HDACi at the indicated doses ( n = 2 biological replicates). ( F ) Histogram indicates the percentage of viable (white), dead (black), or rescued (red) HNT34 cells ( n = 20.000) by the overexpression of PA2G4 on the basis of positivity for annexin V/PI staining after 72 h of HDACis treatment ( n = 2 biological replicates). G Percentage of PA2G4 , EVI1 , and MYC mRNA relative to the control gene ACTB (ΔΔCT) in HNT34 and UCSD/AML1 cells 6 days after shRNA transduction. Data are presented as mean ± SD ( n = 3). Statistical significance was determined by a two-tailed unpaired, parametric t-test. H Proteasome inhibition rescues WS6-induced EVI1 and MYC protein degradation. EVI1, ∆EVI1, MYC expression, and PA2G4 in HNT34 and UCSD/AML1 cell lines after treatment with DMSO and WS6 (24 hr) or MG132 (4 hr) at the indicated concentrations ( n = 2 biological replicates). NT = non-targeting, sh#65 = shRNA directed against PA2G4 in C and F . Source data are provided as a Source Data file. See also Supplementary Fig. - and Supplementary Data .

Article Snippet: The human cell lines MOLM1 (#ACC 720), UCSD/AML1 (#ACC 691), HNT34 (#ACC 600), TF1 (#ACC 334), MUTZ-3 (#ACC 295), 293 T (#ACC 635), OCI/AML3 (#ACC 582), MOLM13 (#ACC 554), NOMO1 (#ACC 542), OCI/AML2 (#ACC 99), GDM1 (#ACC 87), HL-60 (#ACC 3), SKM1 (#ACC 547) and 5637 (#ACC 35) were purchased from the Leibniz-Institut DSMZ-German collection of microorganisms and cell cultures (Germany).

Techniques: Immunoprecipitation, Mass Spectrometry, Gene Expression, Binding Assay, Expressing, shRNA, Transduction, Over Expression, Western Blot, Staining, Control, Two Tailed Test, Inhibition