human myeloid leukemia cell lines k562  (DSMZ)

Journal: bioRxiv

Article Title: Ecological determinants of disease and immunity in myelodysplastic syndromes

doi: 10.64898/2026.05.05.720208

Figure Lengend Snippet: a, Bar plots of relative median fluorescence intensity (MFI) of granzyme B and perforin from intracellular cytokine staining of primary untreated MDS CD8 T cells after exposure to increasing doses of TGFβ. Relative MFI values are shown as mean ± SD. Data from n=6 MDS patients. Mann-Whitney U test used for statistical analysis (*p<0.05, **p<0.01). b, Top, volcano plot of differentially expressed genes in CD8 memory T cells close to (≤20µm) versus far from (>50µm) megakaryocytes. Bottom, schematic of CD8 memory T cells based on their distance to nearest megakaryocytes from spatial data. c, Relative percent spliced in (dPSI) value of differential 3’ splice site in mRNAs from SF3B1 mutant MDS patient bulk RNA-seq versus SF3B1 wild-type MDS. Highlighted names indicate mRNAs encoding proteins involved in TGFβ signaling. d, Diagram of proteins (in green) involved in TGFβ signaling which undergo aberrant RNA splicing in SF3B1 mutant MDS. e, Protein diagram of TGFBR1 with red indicating insertion of four amino acids (GPFS) encoded by the long mRNA isoform promoted in SF3B1 mutant cells. f, Alpha fold model of mutant TGFBR1 (green) overlaid with published crystal structure of wild-type TGFBR1 (gray). The GPFS amino acid insertion seen in SF3B1 mutant cells is indicated in red in the inset. g, Percentage (%) of phospho-SMAD2 Serine 465/467 (pS465/467) in K562 cells with the indicated genetic alterations in SF3B1 or TGFBR1. Mean ± SD. Two-way ANOVA. ***p<0.001, ****p<0.0001. h , Representative flow cytometry histograms of p-SMAD2 S465/467 from (g). i, Schematic of an SF3B1-mutant megakaryocyte with increased TGFβ production suppressing cytotoxic activity of nearby CD8 + T cells (cell-extrinsic effect); mutant cell simultaneously exhibits impaired TGFβ sensing (cell-intrinsic effect).

Article Snippet: The K562 human myeloid leukemia cell line was purchased from American Type Culture Collection (ATCC; #CCL-243).

Techniques: Fluorescence, Staining, MANN-WHITNEY, Mutagenesis, RNA Sequencing, Flow Cytometry, Activity Assay

Journal: bioRxiv

Article Title: Ecological determinants of disease and immunity in myelodysplastic syndromes

doi: 10.64898/2026.05.05.720208

Figure Lengend Snippet: (a) Sashimi plots of bulk RNA-sequencing data of an aberrant 3’ splice site usage in TGFBR1, MAP3K7 and SMURF2 mRNA in SF3B1 mutant acute myeloid leukemia (AML) (top; n=76 patients), SF3B1 wild-type (WT) AML (middle; n=739 patients), and normal bone marrow (bottom; n=26 patients). Red lines indicate SF3B1 mutant-specific junctions while black lines represent junction spanning reads in wild-type cells. The number of reads is listed, and the frequency of reads is in parentheses. b , Crystal structure of the short-isoform of TGFBR1 (grey) overlaid with that of the alpha-fold predicted model of SF3B1 mutant induced long-isoform (green). c, RT-PCR analysis of aberrant 3’ splice events in TGFBR1, MAP3K7, and SMURF2 in human isogenic K562 cells with knockin of SF3B1 K700E mutation. d , RT-PCR analysis of endogenous TGFBR1 , MAP3K7, and SMURF2 splicing in primary samples in healthy bone marrow control patients (n=5), patients with SF3B1 K700E mutant MDS (n=5) and non-splicing factor mutant patients with MDS (n=5). e , Sanger sequencing electropherogram of the top and bottom PCR products from gel-purified TGFBR1 RT-PCRs from MDS SF3B1 mutant patients shown in ( c ). The red box highlights the alternatively spliced sequence in the top band, which includes a 12-nucleotide insertion predominantly observed in SF3B1 K700E mutant MDS patients. The bottom band sequence is displayed below, showing the canonical exonic sequence. f , Western blot of K562 cells with SF3B1 mutation, TGFBR1 knockout (KO), or TGFBR1 KO with addback of TGFBR1 cDNA encoding the long or short isoform. g, Western blot of cytoplasmic, membrane, and soluble nuclear fractions of K562 cells with TGFBR1 KO alone or with overexpression of TGFBR1 cDNA encoding the long or short isoform.

Article Snippet: The K562 human myeloid leukemia cell line was purchased from American Type Culture Collection (ATCC; #CCL-243).

Techniques: RNA Sequencing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Knock-In, Control, Sequencing, Purification, Western Blot, Knock-Out, Membrane, Over Expression

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: BRD4 inhibitor significantly promotes HCP5 super‐enhancer activity and expression. (A) Reverse transcription‐quantitative polymerase chain reaction analysis of HCP5 mRNA expression levels in acute myeloid leukemia cell lines (THP‐1 and HL‐60) treated with the BRD4 inhibitor GNE‐987. (B) Western Blot analysis showing BRD4 expression levels in different groups. (C) Representative agarose gel electrophoresis image and statistical quantification of ChIP‐qPCR products. Compared to the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times. BRD4, Bromodomain‐containing protein 4; HCP5, HLA Complex P5.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Activity Assay, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Agarose Gel Electrophoresis, ChIP-qPCR

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Bromodomain‐containing protein 4 Inhibitor GNE‐987 Significantly Inhibits acute myeloid leukemia Cell Proliferation and Migration and Induces Apoptosis. (A) CCK‐8 assay measuring cell proliferation in each group at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (B) Representative images of Live and Dead staining for each group, with a bar chart depicting the statistical analysis of cell death ratios. Bar = 50 μm. (C) Colony formation assay for each group, with a bar chart showing the statistical analysis of colony numbers. (D) Flow cytometry analysis of apoptosis levels in each group, with a bar chart depicting the statistical analysis of apoptosis rates. Compared with the Ctrl group, * p < 0.05, ** p < 0.01. All cell experiments were repeated three times.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Migration, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: Journal of Cell Communication and Signaling

Article Title: Mechanistic role of GNE‐987 targeting BRD4‐HCP5 axis in pediatric T‐cell acute lymphoblastic leukemia

doi: 10.1002/ccs3.70063

Figure Lengend Snippet: Silencing of HLA Complex P5 Reverses the Inhibitory Effect of GNE‐987 on acute myeloid leukemia Cell Viability. (A) Reverse transcription‐quantitative polymerase chain reaction results verify the silencing efficiency of sh‐HCP5. (B) CCK8 assay measuring cell proliferation at 0, 12, 24, 36, 48, 60, and 72 h, with absorbance detected at OD450. (C) Representative images of Live and Dead staining in each group and a bar chart showing the death ratio; scale bar = 50 μm. (D) Colony formation assay and statistical graph of colony numbers for each group. (E) Flow cytometry analysis of apoptosis levels and a statistical graph of apoptosis rates in each group. For panel A, compared with the sh‐NC group, * p < 0.05, ** p < 0.01. For panels B‐E, compared with the Ctrl group, * p < 0.05, ** p < 0.01; compared with the GNE‐987+sh‐NC group, # p < 0.05, ## p < 0.01. All cell experiments were repeated three times.

Article Snippet: The acute myeloid leukemia (AML) cell lines Tsuchiya Human Phagocyte‐1 (THP‐1) and Human Leukemia‐60 (HL‐60) (TIB‐202 and CCL‐240, ATCC), along with T‐ALL–specific cell lines Jurkat, CCRF‐CEM, MOLT‐4, and RPMI‐8402 (TIB‐152, CCL‐111, CRL‐1582, and CCL‐27, ATCC), were cultured in RPMI‐1640 medium (11875093, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (A5670701, Gibco) and maintained in an incubator at 37°C with 5% CO 2 .

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, CCK-8 Assay, Staining, Colony Assay, Flow Cytometry

Journal: bioRxiv

Article Title: Lineage-restricted dependency on an oncofetal SNHG29 -IGF2BP1 RNA axis in acute megakaryoblastic leukemia

doi: 10.64898/2026.02.07.704501

Figure Lengend Snippet: A Schematic representation of RNA pulldown strategy for identification of SNHG29 -interacting proteins. B Schematic representation of our approach in determining a potentially mechanistic interaction with SNHG29 in M-07e. C RNA pulldown of SNHG29 in M-07e cells. Significantly enriched proteins (Log2 Fold Change > 4, adjusted P < 0.01) highlighted in pink (n=3 biological replicates, t-test, BH multiple testing correction, analysis by Perseus). D Fluorescence-based proliferation assays in M-07e cells with CRISPR-Cas9-mediated IGF2BP1 knockout using 3 sgRNAs. Data normalized to day 0 and respective controls. (mean ± s.e.m; *P<0.05, **P<0.01, ***P<0.001; two-tailed, unpaired t-test) E Dependency scores (DepMap 25Q3) of proteins enriched in SNHG29 pulldown and essential in M-07e (DepMap score < −0.5) across 29 AML cell lines. Specificity Z-scores were calculated across AML cell lines. F Expression of IGF2BP1 in RNA sequencing datasets from fetal liver CD34+ cells (FL CD34, n=5), peripheral blood mobilized CD34+ cells (PB CD34, n=8), AMKL (n=19) and non-megakaryoblastic AML (non-AMKL) (n=130) patient samples. Data shown are Log2 transformed TPM. Box plots show medians, boxes and whiskers according to the Tukey method. ***FDR<0.001 (LIMMA-voom). G eCLIP peak data showing IGF2BP1 binding at the SNHG29 locus in K562. Plus strand peaks with - Log10 P > 4 and Log2 fold change > 3 over size-matched input are displayed. No significant peaks were detected on the minus strand. (n=2) H Gene Set Enrichment Analysis (GSEA) of the top 200 IGF2BP1 eCLIP targets following SNHG29 knockdown in M-07e cells using CRISPRi with two different sgRNAs (left) or two different shRNAs (right) analyzed compared to the respective non-targeting control. Genes from RNA-seq experiments (n=3) were ranked by t-statistic. NES, Normalized Enrichment Score; FDR, False Discovery Rate; ES, Enrichment Score. I GSEA of Hallmark and Reactome gene sets following SNHG29 knockdown via shRNA and CRISPRi (n=3). Gene sets with the highest averaged NES between shRNA and CRISPRi conditions are shown.

Article Snippet: Human myeloid leukemia cell lines K562, THP-1, ML-2, M-07e, KASUMI-1, NOMO-1 and SKNO-1 were procured from DSMZ (Braunschweig, Germany).

Techniques: Fluorescence, CRISPR, Knock-Out, Two Tailed Test, Expressing, RNA Sequencing, Transformation Assay, Binding Assay, Knockdown, Control, shRNA