human acute myeloid leukemia kg1 cell line expressing cd34 surface antigen  (ATCC)

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of differentiation heterogeneity in the OCI-AML8227 cell line model. This cell line produces leukemic blasts expressing immature myeloid markers (CD34) as well as differentiated monocytic markers (CD64 and CD14). CD38 is a transient myeloid differentiation marker, which is initially expressed in early myeloid progenitor cells. b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 96 hours along an 8-point dose curve with venetoclax. Cell viability was assessed by CellTiter Aqueous colorimetric assay. c. AUC values from the dose-response curves for each immunophenotypic population. Significance was evaluated using a two-tailed t-test. d–i. Cell surface expression of CD34, CD38, CD64, and CD14 in OCI-AML8227 cells cultured in triplicate for 72 hours with 1 μM venetoclax or an equivalent volume of DMSO. Cells were analyzed either as immunomagnetically fractionated populations (CD34-enriched and CD34-depleted) or as unfractionated cells. Significance was evaluated using ordinary one-way ANOVA followed by Holm-Šidák post-test correction. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Marker, Cell Culture, Colorimetric Assay, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a, b. OCI-AML8227 cells were immunomagnetically fractionated by CD34 expression and cultured in triplicate for 30 minutes or 6 hours following treatment with 1 μM venetoclax or an equivalent volume of DMSO. Proteins were extracted from cell pellets and either separated by liquid chromatography and analyzed by tandem mass spectrometry for protein identification and quantification or enriched for phosphorylated species prior to LC–MS/MS analysis to profile phosphorylation-dependent signaling. Relative (a) global protein and (b) phosphoprotein abundance at 6 hours following venetoclax treatment in CD34-enriched and CD34-depleted populations were analyzed using proteomic network analysis.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Expressing, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Phospho-proteomics

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Predicted transcription factor activity was evaluated in single-cell RNA-seq data presented in . Significantly enriched transcription factors were identified and denoted as enriched in progenitor (blue), monocytic (gold), or both (green) populations. Gray dots represent transcription factors that did not reach statistical significance (FDR < 0.05). b. Predicted activity of PU.1 visualized by each time point and drug condition. c. Pseudotime values generated in were used to determine each cell’s relative position along the myeloid differentiation trajectory. Predicted activity of PU.1 is visualized along its pseudotime trajectory in each drug condition. d. OCI-AML8227 cells were cultured with DMSO or 1 μM venetoclax and processed at either 0 or 72 hours for single-cell ATAC-seq. Differential peaks were identified from open chromatin regions in each condition. Significant peaks were compared between monocytic and progenitor clusters. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). e, f. Transcription factor motif analysis was performed on (e) upregulated and (f) downregulated regions in monocytic cells relative to progenitor cells as described in panel d. Gray dots represent motifs that did not reach statistical significance (FDR < 0.05). g. Venn diagram displaying the overlap of significantly dysregulated transcription factors identified from single-cell RNA-seq analysis in panel a compared to enriched motifs identified in panels e and f.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Activity Assay, RNA Sequencing, Generated, Cell Culture

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Schematic of the targeted CRISPR screen. A stable Cas9-expressing OCI-AML8227 cell line was generated by electroporation and sorted for GFP expression. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression. Image was created with BioRender. b. Live cell counts determined by forward/side scatter gating, excluding DAPI-stained cells. Counts were normalized to the average of their respective DMSO-treated controls. Blue lines and asterisks indicate comparisons with the safe-harbor locus AAVS1 (control) whereas black lines and asterisks indicate intra-sample comparisons. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. c–h. Live cell counts for each immunophenotypic population, normalized as described in panel b. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. i–j. Representative flow cytometry plots showing CD64-FITC and CD38-APC-Fire surface expression in OCI-AML8227 cells transduced with guide RNAs targeting AAVS1 or SPI1 following treatment with DMSO or 1 μM venetoclax. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: CRISPR, Expressing, Generated, Electroporation, Transduction, Cell Culture, Flow Cytometry, Staining, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. Cells were transduced with three guide RNAs per transcription factor target by electroporation, then cultured in triplicate with DMSO or 1 μM venetoclax for 72 hours before flow cytometry analysis of CD34, CD38, CD64, and CD14 surface expression as described in . Quantification of total cell counts in additional knockout models of OCI-AML8227 cells. Total cell counts were normalized to the average of their respective DMSO-treated controls. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Sensitivity to venetoclax was unchanged by the experimental knockouts compared to control AAVS1 . b–g. Live cell counts for each immunophenotypic population were calculated by multiplying their proportions of live single cells from flow cytometric analysis by their normalized values in panel a. Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. Few changes were observed in knockouts relative to control. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Transduction, Electroporation, Cell Culture, Flow Cytometry, Expressing, Knock-Out, Control

Journal: bioRxiv

Article Title: PU.1 inhibition sensitizes stem-monocytic AML to BCL2 blockade

doi: 10.64898/2026.01.20.700677

Figure Lengend Snippet: a. OCI-AML8227 cells were cultured in triplicate for 72 hours along an 8-point dose curve of DB2313. Cell viability was assessed by CellTiter Aqueous colorimetric assay. b. OCI-AML8227 cells were treated in triplicate with an 8×8 dose matrix of DB2313 and venetoclax for 72 hours prior to viability assessment by CellTiter Aqueous colorimetric assay. Zero interaction potency (ZIP) synergy scores were calculated on the average values for each drug dose. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. c–e. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 0.1 μM venetoclax, 0.5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Live cell counts were determined by forward/side scatter gating and exclusion of DAPI-stained cells, then normalized to DMSO-treated controls. Cells were analyzed by flow cytometry for CD34, CD38, CD64, and CD14 surface expression. Quantification of live cell counts for the remaining cell surface markers is shown in . Significance was evaluated using ordinary two-way ANOVA followed by Holm-Šidák post-test correction. f–h. Live cell counts in OCI-AML8227 cells following 72 hours of treatment with 1 μM venetoclax, 5 μM DB2313, both drugs in combination, or an equivalent volume of DMSO. Analysis was performed as described in panels c–e. i. Transcriptional signatures of nine primary AML samples selected for drug sensitivity evaluation are shown. One sample was excluded from downstream analyses due to widespread cell death (18-00105). j. Primary AML blasts from eight patients with stem-monocytic AML were cultured in triplicate for 72 hours along a 7-point dose curve with venetoclax, DB2313, or equimolar amounts of the drug combination. Viability was assessed using the Guava/EMD Millipore platform after a short incubation with Guava Nexin Reagent (Annexin V–PE + 7-AAD). ZIP synergy scores were calculated from averaged viability data across replicates for each drug dose in primary AML blasts shown in panel i. The white box indicates the DB2313 and venetoclax concentrations corresponding to maximal synergy. k. Venetoclax dose-response curves for each patient at a fixed dose of 1.25 μM DB2313, which corresponds to maximal synergy in panel j. ns = not significant; * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001.

Article Snippet: OCI-AML8227 cells were immunomagnetically fractionated for CD34 surface expression using CD34 Microbeads (Miltenyi Biotec #130-046-702).

Techniques: Cell Culture, Colorimetric Assay, Staining, Flow Cytometry, Expressing, Incubation