Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Schematic of experimental workflow. B) Diagram showing filtering of peptides from mass spectrometry. C) Representative western blot (WB) of lysates isolated from CM + cells, (D) human AML cell lines, or (E) human ALL cell lines immunoprecipitated with anti-RUNX1 or anti-IgG antibodies and blotted for PTBP1. F) Confocal images of Proximity Ligation Assays (PLA) performed in MOLM-13 cells using the indicated antibodies. G) Confocal images of PLAs in MOLM-13 cells treated with vehicle or 10µM entinostat for 48 hours. H) Graph quantifying PLA signal per field (minimum of 50 cells) from cells treated as in G. N>3. * = p < 0.05. **** = p < 0.0001. Scale bar = 20µm.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Mass Spectrometry, Western Blot, Isolation, Immunoprecipitation, Ligation

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Schematic representation of RUNX1 and PTBP1 proteins showing binding domains, and the relevant mutations indicated with arrows. B-D) Representative western blots from HEK293T cells transfected with expression constructs for the indicated proteins, immunoprecipitated (IP’d) and probed with the indicated antibodies. Each experiment was performed at least 3 times.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Construct, Immunoprecipitation

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Violin plot depicting relative PTBP1 expression in human hematopoietic populations and AML subtypes (HSC-Hematopoietic Stem Cell; MPP-Multipotent Progenitor; CMP- Common Myeloid Progenitor; GMP-Granulocyte-Monocyte Progenitor; MEP-Megakaryocyte-Erythrocyte Progenitor; early PM-Early Promyelocyte; late PM-Late Promyelocyte; MY-Myelocyte; MM-Metamyelocytes; BC-Band cell; PMN-Polymorphonuclear cells; Mono-Monocytes). B) Western blot (WB) of PTBP1 protein in healthy bone marrow (HBM) and sorted CD34 + cells from healthy individuals, and primary diagnosis and relapse (rel) AML samples of the indicated FAB stage. C) Representative confocal images and D) graph of PLA intensity/field (minimum of 50 cells) in mouse CM + leukemia cells and WT lineage depleted (Lin-) cells. E) Representative confocal images of PLA performed with RUNX1 and PTBP1 antibodies in healthy human CD34+ cells and the human AML patient sample 6406. N=3. **** = p < 0.0001. Scale bar - 20µm.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Expressing, Western Blot, Biomarker Discovery

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Heatmaps and mean signal intensities from PTBP1 and RUNX1 CUT&Tag, centered on the peak, with 0 indicating the midpoint (±1kb) grouped into regions with PTBP1 high (PTPB1>RUNX1), PTBP1 and RUNX1 high (PTBP1 & RUNX1), and RUNX1 high (PTBP1

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Generated

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Principal component analysis of MOLM-13 sh SCR (SC) and sh PTBP1 (KD) cells from long-read RNA sequencing data. B) Pie chart revealing altered splicing events in response to PTBP1 KD in MOLM-13s (Alt 3’ SS – Alternative 3’ Splice Site; Alt 5’ SS – Alternate 5’ Splice Site; Alt TSS – Alternate Transcription Start Site; Alt TTS – Alternate Transcription Termination Site; SE – Skipped Exon; RI – Retained Intron; MEE – Mutually Exclusive Exon; MES – Multiple Exon Skipping). C) Analysis of the splicing events in (B.) revealing genes with differential isoform expression in response to PTBP1 KD in MOLM-13s. D) Volcano plot showing differentially expressed genes in response to PTBP1 KD in MOLM-13s. Highlighted genes include Inhibitor of Differentiation 1 ( ID1 ), Hexokinase-2 ( HK2 ), PTBP1 , and PTBP2 . E) Analysis of differentially expressed genes by EnrichR showing top KEGG pathways affected after PTBP1 KD. F) Integrated Genomics Viewer (IGV) tracks depicting the binding of RUNX1 and PTBP1 at HK2 and ID1 gene loci. N≥3.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: RNA Sequencing, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Growth curve of MOLM-13 sh SCR and sh PTBP1 cells. B) Growth curve of Kasumi-1 shS CR and sh PTBP1 cells. C) Representative plots and quantification from flow cytometry analysis of CD15 and CD11b of MOLM-13 sh SCR and sh PTBP1 cells and D) Kasumi-1 sh SCR and sh PTBP1 cells. E) Representative plots and quantification of Annexin V+ cells in MOLM-13 sh SCR and sh PTBP1 cells on day 7 of doxycycline induction. F) Representative plots and quantification of flow cytometry analysis of Annexin V and DAPI on day 21 of doxycycline induction in MOLM-13 sh SCR and sh PTBP1 cells and G) Kasumi-1 sh SCR and sh PTBP1 cells. N≥3. * = p < 0.05, ** = p < 0.01. **** = p < 0.0001.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Representative WB and quantification of relative abundance of Inhibitor of DNA binding 1 (ID1) at day 7 of PTBP1 KD in MOLM-13s. B) Representative WB and quantification of relative abundance of Hexokinase-2 (HK2) at day 7 of PTBP1 KD in MOLM-13s. Dashed line indicates non-adjacent lanes from the same gel. C) Quantification of lactate from lactate release assays in MOLM-13 sh SCR and sh PTBP1 cells at day 7 of KD. D) Representative WB and quantification of relative abundance of Glucose transporter-1 (GLUT1) on day 14 of PTBP1 KD in MOLM-13s. E) Quantification of mean fluorescence intensity (MFI) from glucose uptake assays on day 14 of doxycycline treatment in MOLM-13 sh SCR and sh PTBP1 cells. F) Representative WB and quantification of relative abundance of HK2 And G) GLUT1 in MOLM 13 cells treated with 5 µM Entinostat (ENT) or DMSO for 48 hours. N≥3. * p ≤ 0.05. ** = p < 0.01.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Binding Assay, Fluorescence

Journal: bioRxiv

Article Title: The Splicing Factor PTBP1 interacts with RUNX1 and is Required for Leukemia Cell Survival

doi: 10.1101/2025.05.16.654547

Figure Lengend Snippet: A) Representative WB and B) quantification of (S139) phosphorylated ɣH2A.X (S139) levels after 7 days of PTBP1 KD in MOLM-13 cells. C) Representative plots of flow cytometry analysis and D) quantification of Annexin V and DAPI staining in MOLM-13 sh SCR and sh PTBP1 cells treated with 5µM cytarabine for 72 hours after 7 days of PTBP1 KD. N≥3. *** = p < 0.001.

Article Snippet: The Myc tag WT PTBP1 construct was purchased from Addgene (Plasmid# 23024).

Techniques: Flow Cytometry, Staining