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ATCC human chronic myeloid leukemia cell line k562

Results from <t>K562/luc</t> (expressing shNEG, Firefly luciferase, and GFP) alone (mono) or mixed with human primary bone marrow mesenchymal stem cells (with hMSC) were subcutaneously injected in mice to form xenografts. (A) Scheme demonstrating the experimental setup; samples were collected 21 days after subcutaneous injection of cells in mice. (B) Weight of xenografts formed by K562/luc mono or a mix with hMSCs in mice (n=5 in each variant), and the two-tailed, nonparametric Mann-Whitney test was used to check the difference significance; the exact significance ( P ) values are presented in the plot. (C-H) Short-read sequencing RNA analysis results of samples from cells isolated from xenografts mono and with hMSC treated for the last 14 days with vehicle (shNEG) by the fluorescence-activated cell sorting (FACS) with BD Aria. (C) Representative scatterplots (n=3) with the gating strategy used for the FACS-sorting of live xenograft cells (negative for Viability dye-eFluor780 staining), expressing GFP (GFP+) and stained positive with the antibody against human CD45 with BV421 fluorophore (hCD45+). (D-G) Results of differential expression analysis of the variant shNEG versus mono by DeSeq2 of RNA sequenced from FACS-sorted cells. Presented are the results from cells isolated from three different xenografts for each variant. (D) Principal component analysis. (E) Number of genes with fold change absolute value ≥ 50% and p-value corrected for multiple testing using the Benjamini-Hochberg P adj. ≤ 0.05, that are upregulated (UP) or downregulated (DOWN) in cells from xenograft (Xgraft) shNEG versus mono. (F) Expression level (counts corrected for the sample sequencing depth) in each xenograft (n=3) of the top 150 genes (each row) with the most significant (P adj. ) fold change in expression level. (G) The top of Gene Ontology Biological Processes (GOBP) terms from the Gene Set Enrichment Analysis (GSEA) of the DeSeq2 results, with the highest positive (left) and negative (right) normalized enrichment score (NES), P adj. ≤ 0.05 (FDR) and number of genes in the sample annotated per term (size) > 20. The running enrichment score is presented by a grey line for each of the ranked genes marked with a vertical black line. (H) The Reactome terms identified by the GSEA of the UP or DOWN genes in the variant shNEG versus mono, with NES ≥ 1.5 (absolute value), p value ≤ 0.05, and size > 30.

Human Chronic Myeloid Leukemia Cell Line K562, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 10959 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma"

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

Journal: bioRxiv

doi: 10.64898/2026.05.29.728710

Results from K562/luc (expressing shNEG, Firefly luciferase, and GFP) alone (mono) or mixed with human primary bone marrow mesenchymal stem cells (with hMSC) were subcutaneously injected in mice to form xenografts. (A) Scheme demonstrating the experimental setup; samples were collected 21 days after subcutaneous injection of cells in mice. (B) Weight of xenografts formed by K562/luc mono or a mix with hMSCs in mice (n=5 in each variant), and the two-tailed, nonparametric Mann-Whitney test was used to check the difference significance; the exact significance ( P ) values are presented in the plot. (C-H) Short-read sequencing RNA analysis results of samples from cells isolated from xenografts mono and with hMSC treated for the last 14 days with vehicle (shNEG) by the fluorescence-activated cell sorting (FACS) with BD Aria. (C) Representative scatterplots (n=3) with the gating strategy used for the FACS-sorting of live xenograft cells (negative for Viability dye-eFluor780 staining), expressing GFP (GFP+) and stained positive with the antibody against human CD45 with BV421 fluorophore (hCD45+). (D-G) Results of differential expression analysis of the variant shNEG versus mono by DeSeq2 of RNA sequenced from FACS-sorted cells. Presented are the results from cells isolated from three different xenografts for each variant. (D) Principal component analysis. (E) Number of genes with fold change absolute value ≥ 50% and p-value corrected for multiple testing using the Benjamini-Hochberg P adj. ≤ 0.05, that are upregulated (UP) or downregulated (DOWN) in cells from xenograft (Xgraft) shNEG versus mono. (F) Expression level (counts corrected for the sample sequencing depth) in each xenograft (n=3) of the top 150 genes (each row) with the most significant (P adj. ) fold change in expression level. (G) The top of Gene Ontology Biological Processes (GOBP) terms from the Gene Set Enrichment Analysis (GSEA) of the DeSeq2 results, with the highest positive (left) and negative (right) normalized enrichment score (NES), P adj. ≤ 0.05 (FDR) and number of genes in the sample annotated per term (size) > 20. The running enrichment score is presented by a grey line for each of the ranked genes marked with a vertical black line. (H) The Reactome terms identified by the GSEA of the UP or DOWN genes in the variant shNEG versus mono, with NES ≥ 1.5 (absolute value), p value ≤ 0.05, and size > 30.

Figure Legend Snippet: Results from K562/luc (expressing shNEG, Firefly luciferase, and GFP) alone (mono) or mixed with human primary bone marrow mesenchymal stem cells (with hMSC) were subcutaneously injected in mice to form xenografts. (A) Scheme demonstrating the experimental setup; samples were collected 21 days after subcutaneous injection of cells in mice. (B) Weight of xenografts formed by K562/luc mono or a mix with hMSCs in mice (n=5 in each variant), and the two-tailed, nonparametric Mann-Whitney test was used to check the difference significance; the exact significance ( P ) values are presented in the plot. (C-H) Short-read sequencing RNA analysis results of samples from cells isolated from xenografts mono and with hMSC treated for the last 14 days with vehicle (shNEG) by the fluorescence-activated cell sorting (FACS) with BD Aria. (C) Representative scatterplots (n=3) with the gating strategy used for the FACS-sorting of live xenograft cells (negative for Viability dye-eFluor780 staining), expressing GFP (GFP+) and stained positive with the antibody against human CD45 with BV421 fluorophore (hCD45+). (D-G) Results of differential expression analysis of the variant shNEG versus mono by DeSeq2 of RNA sequenced from FACS-sorted cells. Presented are the results from cells isolated from three different xenografts for each variant. (D) Principal component analysis. (E) Number of genes with fold change absolute value ≥ 50% and p-value corrected for multiple testing using the Benjamini-Hochberg P adj. ≤ 0.05, that are upregulated (UP) or downregulated (DOWN) in cells from xenograft (Xgraft) shNEG versus mono. (F) Expression level (counts corrected for the sample sequencing depth) in each xenograft (n=3) of the top 150 genes (each row) with the most significant (P adj. ) fold change in expression level. (G) The top of Gene Ontology Biological Processes (GOBP) terms from the Gene Set Enrichment Analysis (GSEA) of the DeSeq2 results, with the highest positive (left) and negative (right) normalized enrichment score (NES), P adj. ≤ 0.05 (FDR) and number of genes in the sample annotated per term (size) > 20. The running enrichment score is presented by a grey line for each of the ranked genes marked with a vertical black line. (H) The Reactome terms identified by the GSEA of the UP or DOWN genes in the variant shNEG versus mono, with NES ≥ 1.5 (absolute value), p value ≤ 0.05, and size > 30.

Techniques Used: Expressing, Luciferase, Injection, Variant Assay, Two Tailed Test, MANN-WHITNEY, Sequencing, Isolation, Fluorescence, FACS, Staining, Quantitative Proteomics

(A) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days, when the GFP and human CD45 positive cells were FACS-sorted from xenografts for RNA isolation and sequencing. (B) Number of intersecting genes (compared groups indicated by black dot) that at the RNA level are upregulated (UP; log2 value of fold change ≥ 0.6) or downregulated (DOWN; log2 value of fold change ≤ -0.6) in shNEG cells xenografts imatinib versus vehicle treated and in CD34 positive cells from bone marrow biopsies of a patient with CML (data deposited at GEO under accession number GSE310243; from ) obtained after 6 months of imatinib therapy (combined samples SRR36072325 and SRR36072321) versus obtained at the diagnosis (combined SRR36072320 and SRR36072324). (B-D) Yellow shadow marks a group of genes that are UP or DOWN upon imatinib treatment in shNEG and CML cells (shNEG&CML), selected for subsequent analysis in (C) and (D). (C) Reactome terms identified by the GSEA analysis of the UP or DOWN genes in shNEG&CML with normalized enrichment score ≥ 1.5 (absolute value), p value ≤ 0.05, and number of genes in the sample annotated per term (size) ≥ 15. (C-D) The Reactome leading-edge genes identified by GSEA, annotated to terms marked with a brown triangle in (C) were selected to compare their expression level in single-cell RNA-seq data in (D) from bone marrow biopsies of healthy donors and CML patients with different responses to imatinib therapy. (D) Analysis of data at the Single-cell atlas of diagnostic Chronic Myeloid Leukemia bone marrow (scdbm) for CD34+ cells subtype . Data and detailed description of patient classification available: http://scdbm.ddnetbio.com ; A – responded to IM within 12 months; B – IM treatment failed within 18 months; C – resistant to IM and other TKI-s.

Figure Legend Snippet: (A) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days, when the GFP and human CD45 positive cells were FACS-sorted from xenografts for RNA isolation and sequencing. (B) Number of intersecting genes (compared groups indicated by black dot) that at the RNA level are upregulated (UP; log2 value of fold change ≥ 0.6) or downregulated (DOWN; log2 value of fold change ≤ -0.6) in shNEG cells xenografts imatinib versus vehicle treated and in CD34 positive cells from bone marrow biopsies of a patient with CML (data deposited at GEO under accession number GSE310243; from ) obtained after 6 months of imatinib therapy (combined samples SRR36072325 and SRR36072321) versus obtained at the diagnosis (combined SRR36072320 and SRR36072324). (B-D) Yellow shadow marks a group of genes that are UP or DOWN upon imatinib treatment in shNEG and CML cells (shNEG&CML), selected for subsequent analysis in (C) and (D). (C) Reactome terms identified by the GSEA analysis of the UP or DOWN genes in shNEG&CML with normalized enrichment score ≥ 1.5 (absolute value), p value ≤ 0.05, and number of genes in the sample annotated per term (size) ≥ 15. (C-D) The Reactome leading-edge genes identified by GSEA, annotated to terms marked with a brown triangle in (C) were selected to compare their expression level in single-cell RNA-seq data in (D) from bone marrow biopsies of healthy donors and CML patients with different responses to imatinib therapy. (D) Analysis of data at the Single-cell atlas of diagnostic Chronic Myeloid Leukemia bone marrow (scdbm) for CD34+ cells subtype . Data and detailed description of patient classification available: http://scdbm.ddnetbio.com ; A – responded to IM within 12 months; B – IM treatment failed within 18 months; C – resistant to IM and other TKI-s.

Techniques Used: Expressing, Luciferase, Injection, Isolation, Sequencing, Biomarker Discovery, Single Cell, RNA Sequencing, Diagnostic Assay

(A) Colony formation by K562 cells expressing shRNA non-targeting (shNEG) or targeting mRNA of TIAL1 (shTIAR), TIA1 (shTIA-1), or FMR1 (shFMRP) that were collected from ex vivo hypoxic (1.5% O2) co-culture with HS-5 bone marrow stromal fibroblasts and treated with 1 μM imatinib (IM) or 50nM Talazoparib (BMN) added in two doses following the experimental scheme (left panel). Number of colonies in each of the 3 technical replicates from 3-4 independent biological experiments presented as % change relative to the untreated cells (dashed black line) set as 100 %. Student’s two-tailed t-test was used to compare two samples marked by the black line; #### or **** - p<0.0001, ns - p > 0.05. (B) Scheme explaining the experimental setup based on the subcutaneous implantation in mice of 3D printed scaffolds (photo taken with a Samsung mobile phone camera) seeded with human cells differentiated into osteoblasts and K562/luc cells with shNEG, shTIAR or shTIA-1, followed by IM treatment for 14 days. (C) Bioluminescence signal monitored in mice after 2 weeks of IM or vehicle treatment (timeline explained in (B) ) following luciferin injection, collected in the Burker’s Xtreme In-Vivo chamber for 30 sec, and overlaid on the mouse X-ray image. Scale presents the signal intensity of the color coding. (D) Sum of the signal intensity (P) collected per second (s) and area (mm 2 ) for each mouse analyzed (single dot) is presented. (E) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG or shTIAR, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days. (F) Weight of each xenograft isolated from mice (single dot) formed by K562/luc cells with shNEG or shTIAR (as in (E) ) presented as fold change of the weight mean value of xenografts from mice treated with vehicle; mean value indicated with the black line. The nonparametric two-tailed Mann-Whitney test was used for comparisons indicated by black lines underneath the exact significance ( P ) values are presented in the plot. (A,D,F) Two-way Anova was used to compare shTIAR upon IM versus other variants; @ - P < 0.002.

Figure Legend Snippet: (A) Colony formation by K562 cells expressing shRNA non-targeting (shNEG) or targeting mRNA of TIAL1 (shTIAR), TIA1 (shTIA-1), or FMR1 (shFMRP) that were collected from ex vivo hypoxic (1.5% O2) co-culture with HS-5 bone marrow stromal fibroblasts and treated with 1 μM imatinib (IM) or 50nM Talazoparib (BMN) added in two doses following the experimental scheme (left panel). Number of colonies in each of the 3 technical replicates from 3-4 independent biological experiments presented as % change relative to the untreated cells (dashed black line) set as 100 %. Student’s two-tailed t-test was used to compare two samples marked by the black line; #### or **** - p<0.0001, ns - p > 0.05. (B) Scheme explaining the experimental setup based on the subcutaneous implantation in mice of 3D printed scaffolds (photo taken with a Samsung mobile phone camera) seeded with human cells differentiated into osteoblasts and K562/luc cells with shNEG, shTIAR or shTIA-1, followed by IM treatment for 14 days. (C) Bioluminescence signal monitored in mice after 2 weeks of IM or vehicle treatment (timeline explained in (B) ) following luciferin injection, collected in the Burker’s Xtreme In-Vivo chamber for 30 sec, and overlaid on the mouse X-ray image. Scale presents the signal intensity of the color coding. (D) Sum of the signal intensity (P) collected per second (s) and area (mm 2 ) for each mouse analyzed (single dot) is presented. (E) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG or shTIAR, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days. (F) Weight of each xenograft isolated from mice (single dot) formed by K562/luc cells with shNEG or shTIAR (as in (E) ) presented as fold change of the weight mean value of xenografts from mice treated with vehicle; mean value indicated with the black line. The nonparametric two-tailed Mann-Whitney test was used for comparisons indicated by black lines underneath the exact significance ( P ) values are presented in the plot. (A,D,F) Two-way Anova was used to compare shTIAR upon IM versus other variants; @ - P < 0.002.

Techniques Used: Expressing, shRNA, Ex Vivo, Co-Culture Assay, Two Tailed Test, Injection, In Vivo, Luciferase, Isolation, MANN-WHITNEY

(A) Comparison of gene expression in K562/luc cells with shNEG or shTIAR, FACS-sorted from xenografts (Xgraft), with level upon 2 weeks of IM versus vehicle (C) treatment changed with log2 value of fold change (Log2FC) ≥ 0.6 (UP) or ≤ -0.6 (DOWN); reversed regulation in shTIAR cells in purple. (B) Gene Ontology Molecular Function terms identified by the GSEA of genes that upon IM vs C in Xgraft of shNEG are reversely regulated in shTIAR (shaded in purple). In grey circles - UP (Log2FC > 0.6) n = 1993, brown circles DOWN (Log2FC < -0.6) n = 1756. Selected terms with NESabs ≥ 1.45, p-value ≤ 0.05, and the number of genes in the sample annotated per term (size) ≥ 10; n -number of genes. (C) Proportion of alternative splicing events (AS) changed upon shTIAR versus shNEG in cells from xenografts treated with vehicle (C) or imatinib (IM) for 2 weeks. Significant events from rMATS analysis requiring at least 20 reads/event, absolute change in PSI (Percent Spliced In) > 0.1 with FDR ≤ 0.05; total number in 3 experiments (n) above the bar. (D) Comparison of intron retention (RI; left panel) and cassette exon (CE; right panel) AS with significant PSI changed in shTIAR versus shNEG in cells from Xgraft or CO treated with IM. (E) Change in expression level of genes in Xgraft IM versus vehicle-treated for a subset of genes with significant changes shTIAR vs shNEG in RI (left panel) or SE (right panel) in cells from Xgraft and CO treated with IM; Log2FC ≤ -0.6 in brown, ≥ 0.6 in grey. (F) Difference in PSI value of CE, RI, and mutually exclusive exons (MXE) AS in: moro – shTIAR versus shNEG xenograft cells from mice treated with IM (Xgraft_IM); yellow – CD34 + enriched cells from bone marrow biopsies of a CML patient collected at 6-month IM therapy versus diagnosis GSE310243 (see in ). Included only with PSI > 0.1 and with FDR ≤ 0.05. (G) Expression level of genes with changes in AS (selected in (E) and (F) ) analyzed at the scdbm for the CD34 + cells subtype. Patient classification in .

Figure Legend Snippet: (A) Comparison of gene expression in K562/luc cells with shNEG or shTIAR, FACS-sorted from xenografts (Xgraft), with level upon 2 weeks of IM versus vehicle (C) treatment changed with log2 value of fold change (Log2FC) ≥ 0.6 (UP) or ≤ -0.6 (DOWN); reversed regulation in shTIAR cells in purple. (B) Gene Ontology Molecular Function terms identified by the GSEA of genes that upon IM vs C in Xgraft of shNEG are reversely regulated in shTIAR (shaded in purple). In grey circles - UP (Log2FC > 0.6) n = 1993, brown circles DOWN (Log2FC < -0.6) n = 1756. Selected terms with NESabs ≥ 1.45, p-value ≤ 0.05, and the number of genes in the sample annotated per term (size) ≥ 10; n -number of genes. (C) Proportion of alternative splicing events (AS) changed upon shTIAR versus shNEG in cells from xenografts treated with vehicle (C) or imatinib (IM) for 2 weeks. Significant events from rMATS analysis requiring at least 20 reads/event, absolute change in PSI (Percent Spliced In) > 0.1 with FDR ≤ 0.05; total number in 3 experiments (n) above the bar. (D) Comparison of intron retention (RI; left panel) and cassette exon (CE; right panel) AS with significant PSI changed in shTIAR versus shNEG in cells from Xgraft or CO treated with IM. (E) Change in expression level of genes in Xgraft IM versus vehicle-treated for a subset of genes with significant changes shTIAR vs shNEG in RI (left panel) or SE (right panel) in cells from Xgraft and CO treated with IM; Log2FC ≤ -0.6 in brown, ≥ 0.6 in grey. (F) Difference in PSI value of CE, RI, and mutually exclusive exons (MXE) AS in: moro – shTIAR versus shNEG xenograft cells from mice treated with IM (Xgraft_IM); yellow – CD34 + enriched cells from bone marrow biopsies of a CML patient collected at 6-month IM therapy versus diagnosis GSE310243 (see in ). Included only with PSI > 0.1 and with FDR ≤ 0.05. (G) Expression level of genes with changes in AS (selected in (E) and (F) ) analyzed at the scdbm for the CD34 + cells subtype. Patient classification in .

Techniques Used: Comparison, Gene Expression, Alternative Splicing, Expressing, Biomarker Discovery

(A) Scheme explaining experimental setup to determine proteomic changes using the quantitative BONCAT (QuaNCAT). K562 cells expressing shNEG or shTIAR growing in co-culture with HS-5 cells under hypoxia (1.5% O2) for 1.5 days were treated with imatinib (IM) for 18h before the bioorthogonal noncanonical amino acid tagging (BONCAT) of nascent proteins synthesized in cells for 4h. (B) Upper panel - number of genes and nascent proteins showing increased (UP; log₂ fold change [Log₂FC] ≥ 0.6) or decreased (DOWN; Log₂FC ≤ −0.6) abundance in IM-treated shTIAR cells compared with shNEG cells, with a significance threshold of p ≤ 0.05. In total, 2186 nascent proteins were quantified in the BONCAT experiment. Lower panel - scatter plot showing intensity-based absolute quantification (iBAQ), used as an estimate of relative protein molar abundance, plotted against Log₂FC values for BONCAT-identified proteins in shTIAR versus shNEG cells. Purple dots indicate hits with significant changes at both the protein and mRNA levels. Dashed vertical lines mark the Log₂FC thresholds of −0.6 and 0.6. (C) Functional annotation Gene Ontology Molecular Function enrichment analysis of UP (brown) or DOWN (purple) proteins with ClueGO/CytoScape, displaying proteins annotated with the term; only one side enriched terms with FDR < 0.05. (D) Lower panel - number of proteins that upon shTIAR are UP and DOWN regulated, for which mRNA was detected in the RNA immunoprecipitated (IP) in TIAR protein complexes (RIP), enriched in samples obtained with anti-TIAR antibody versus the same isotype non-binding antibody (ISO) (n=3647). Upper panel - example Image of Western blotting analysis of IP, with a sample of cell lysate used for IP loaded for reference (input). (E) Changes in the mRNA level, determined by real-time PCR and quantified using the ddCT method, expressed as log2 of change between IM-treated versus untreated cells, detected in samples from whole cells (upper panel) and anti-TIAR RIP (lower panel). Mean of 3 independent experiments with ± range is presented. Student’s t-test two-way was used to compare the difference between IM to C; * p ≤ 0.05, ** p≤0.005. (F) Number of genes identified in anti-TIAR RIP that show significant changes in intron retention (RI) or cassette exon alternative splicing (CE) in shTIAR versus shNEG alternative splicing analysis of RNA from IM-treated cells. Number of genes in the intersection provided above the bar; comparisons indicated by the black dot. (G) Sashimi plot (middle panel) demonstrating splicing of EIF4A2 mRNA within the region encompassing exons 8-11 and 3’UTR in RNA from anti-TIAR RIP. Alternative splice site usage marked by the purple line, and the percent usage ± ME (n=3) in numbers by the lines, reads coverage from 0-145 shown in grey, alternatively spliced exon marked by shaded yellow. Gene region scheme in the top. (H) Percent transcripts with TIAR-dependent alternative exon inclusion in K562 cells from xenografts treated with IM. Student’s t-test was used to compare results from three experiments (each dot) for the mean value marked with a thick black line; * p = 0.021.

Figure Legend Snippet: (A) Scheme explaining experimental setup to determine proteomic changes using the quantitative BONCAT (QuaNCAT). K562 cells expressing shNEG or shTIAR growing in co-culture with HS-5 cells under hypoxia (1.5% O2) for 1.5 days were treated with imatinib (IM) for 18h before the bioorthogonal noncanonical amino acid tagging (BONCAT) of nascent proteins synthesized in cells for 4h. (B) Upper panel - number of genes and nascent proteins showing increased (UP; log₂ fold change [Log₂FC] ≥ 0.6) or decreased (DOWN; Log₂FC ≤ −0.6) abundance in IM-treated shTIAR cells compared with shNEG cells, with a significance threshold of p ≤ 0.05. In total, 2186 nascent proteins were quantified in the BONCAT experiment. Lower panel - scatter plot showing intensity-based absolute quantification (iBAQ), used as an estimate of relative protein molar abundance, plotted against Log₂FC values for BONCAT-identified proteins in shTIAR versus shNEG cells. Purple dots indicate hits with significant changes at both the protein and mRNA levels. Dashed vertical lines mark the Log₂FC thresholds of −0.6 and 0.6. (C) Functional annotation Gene Ontology Molecular Function enrichment analysis of UP (brown) or DOWN (purple) proteins with ClueGO/CytoScape, displaying proteins annotated with the term; only one side enriched terms with FDR < 0.05. (D) Lower panel - number of proteins that upon shTIAR are UP and DOWN regulated, for which mRNA was detected in the RNA immunoprecipitated (IP) in TIAR protein complexes (RIP), enriched in samples obtained with anti-TIAR antibody versus the same isotype non-binding antibody (ISO) (n=3647). Upper panel - example Image of Western blotting analysis of IP, with a sample of cell lysate used for IP loaded for reference (input). (E) Changes in the mRNA level, determined by real-time PCR and quantified using the ddCT method, expressed as log2 of change between IM-treated versus untreated cells, detected in samples from whole cells (upper panel) and anti-TIAR RIP (lower panel). Mean of 3 independent experiments with ± range is presented. Student’s t-test two-way was used to compare the difference between IM to C; * p ≤ 0.05, ** p≤0.005. (F) Number of genes identified in anti-TIAR RIP that show significant changes in intron retention (RI) or cassette exon alternative splicing (CE) in shTIAR versus shNEG alternative splicing analysis of RNA from IM-treated cells. Number of genes in the intersection provided above the bar; comparisons indicated by the black dot. (G) Sashimi plot (middle panel) demonstrating splicing of EIF4A2 mRNA within the region encompassing exons 8-11 and 3’UTR in RNA from anti-TIAR RIP. Alternative splice site usage marked by the purple line, and the percent usage ± ME (n=3) in numbers by the lines, reads coverage from 0-145 shown in grey, alternatively spliced exon marked by shaded yellow. Gene region scheme in the top. (H) Percent transcripts with TIAR-dependent alternative exon inclusion in K562 cells from xenografts treated with IM. Student’s t-test was used to compare results from three experiments (each dot) for the mean value marked with a thick black line; * p = 0.021.

Techniques Used: Expressing, Co-Culture Assay, Synthesized, Quantitative Proteomics, Functional Assay, Immunoprecipitation, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Alternative Splicing

$(A) Expression level of genes analyzed at the scdbm for CD34 + cells subtype; patient classification explained in . (B-H) Impact of shTIAR compared to shNEG analyzed in K562 cells that were co-cultured with HS-5 cells under hypoxia (1.5%O2) 2 days before initiation of 18h culture with imatinib (IM) treatment or without (C). (B) Protein level in whole cell extracts analyzed by Western blot, representative images of immunoblots (n=3) presented. (C) Mitochondrial (mit.) membrane potential measured using JC-1 probe; values for signal from probe aggregates (red) in polarized mitochondria expressed as % of signal from the probe in the cells (n=3), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a control. (C,D,F,H) Bars represent the mean of values from independent experiments (indicated by dots) with ± SD. Student’s t-test was used to determine the significance of the difference shTIAR vs shNEG (#) and IM vs C (*); * - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001 (D) Lipid peroxidation measured with click-it chemistry by flow cytometry. Fluorescence intensity GeoMean expressed as fold change of value in the untreated shNEG cells (n=5). (E-H) Presentation on the cluster of cell differentiation (CD) surface protein markers CD45 and CD235a on K562 cells with shNEG or shTIAR isolated from co-culture established in hypoxia (1.5%O2) a day before initiation of treatment with IM for 48h (E-G) or FACS-sorted K562/luc GFP positive cells from xenografts (H) . (E-F,H) Percentage of parental live cell subpopulations that are: double positive for CD45 and CD235a (CD235a&CD45), positive only for CD45, or only for CD235a, or negative for both CD markers. Representative scatter plots in (E) , summary of independent co-culture experiments (n=4) in (F) . (G) Fluorescence intensity of surface CD36 protein staining in the fraction of cells positive for CD235a or CD45. Numbers correspond to fold change in shTIAR to shNEG ± ME (n=2). (H) Percent GFP and hCD45-positive cells from different xenografts (n=4) that are positive for CD235a. Two-way ANOVA test was used to for comparison of shNEG_C to other variants (@), and p=0.0046.$
Figure Legend Snippet: (A) Expression level of genes analyzed at the scdbm for CD34 + cells subtype; patient classification explained in . (B-H) Impact of shTIAR compared to shNEG analyzed in K562 cells that were co-cultured with HS-5 cells under hypoxia (1.5%O2) 2 days before initiation of 18h culture with imatinib (IM) treatment or without (C). (B) Protein level in whole cell extracts analyzed by Western blot, representative images of immunoblots (n=3) presented. (C) Mitochondrial (mit.) membrane potential measured using JC-1 probe; values for signal from probe aggregates (red) in polarized mitochondria expressed as % of signal from the probe in the cells (n=3), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a control. (C,D,F,H) Bars represent the mean of values from independent experiments (indicated by dots) with ± SD. Student’s t-test was used to determine the significance of the difference shTIAR vs shNEG (#) and IM vs C (*); * - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001 (D) Lipid peroxidation measured with click-it chemistry by flow cytometry. Fluorescence intensity GeoMean expressed as fold change of value in the untreated shNEG cells (n=5). (E-H) Presentation on the cluster of cell differentiation (CD) surface protein markers CD45 and CD235a on K562 cells with shNEG or shTIAR isolated from co-culture established in hypoxia (1.5%O2) a day before initiation of treatment with IM for 48h (E-G) or FACS-sorted K562/luc GFP positive cells from xenografts (H) . (E-F,H) Percentage of parental live cell subpopulations that are: double positive for CD45 and CD235a (CD235a&CD45), positive only for CD45, or only for CD235a, or negative for both CD markers. Representative scatter plots in (E) , summary of independent co-culture experiments (n=4) in (F) . (G) Fluorescence intensity of surface CD36 protein staining in the fraction of cells positive for CD235a or CD45. Numbers correspond to fold change in shTIAR to shNEG ± ME (n=2). (H) Percent GFP and hCD45-positive cells from different xenografts (n=4) that are positive for CD235a. Two-way ANOVA test was used to for comparison of shNEG_C to other variants (@), and p=0.0046.

Techniques Used: Expressing, Cell Culture, Western Blot, Membrane, Control, Flow Cytometry, Fluorescence, Cell Differentiation, Isolation, Co-Culture Assay, Staining, Comparison

Cell Culture:

Article Title: Low-Dose Treatment with Epirubicin, a Novel Histone Deacetylase 1 Inhibitor, Exerts Anti-leukemic Effects by Inducing Ferroptosis.
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Image Search Results

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: Results from K562/luc (expressing shNEG, Firefly luciferase, and GFP) alone (mono) or mixed with human primary bone marrow mesenchymal stem cells (with hMSC) were subcutaneously injected in mice to form xenografts. (A) Scheme demonstrating the experimental setup; samples were collected 21 days after subcutaneous injection of cells in mice. (B) Weight of xenografts formed by K562/luc mono or a mix with hMSCs in mice (n=5 in each variant), and the two-tailed, nonparametric Mann-Whitney test was used to check the difference significance; the exact significance ( P ) values are presented in the plot. (C-H) Short-read sequencing RNA analysis results of samples from cells isolated from xenografts mono and with hMSC treated for the last 14 days with vehicle (shNEG) by the fluorescence-activated cell sorting (FACS) with BD Aria. (C) Representative scatterplots (n=3) with the gating strategy used for the FACS-sorting of live xenograft cells (negative for Viability dye-eFluor780 staining), expressing GFP (GFP+) and stained positive with the antibody against human CD45 with BV421 fluorophore (hCD45+). (D-G) Results of differential expression analysis of the variant shNEG versus mono by DeSeq2 of RNA sequenced from FACS-sorted cells. Presented are the results from cells isolated from three different xenografts for each variant. (D) Principal component analysis. (E) Number of genes with fold change absolute value ≥ 50% and p-value corrected for multiple testing using the Benjamini-Hochberg P adj. ≤ 0.05, that are upregulated (UP) or downregulated (DOWN) in cells from xenograft (Xgraft) shNEG versus mono. (F) Expression level (counts corrected for the sample sequencing depth) in each xenograft (n=3) of the top 150 genes (each row) with the most significant (P adj. ) fold change in expression level. (G) The top of Gene Ontology Biological Processes (GOBP) terms from the Gene Set Enrichment Analysis (GSEA) of the DeSeq2 results, with the highest positive (left) and negative (right) normalized enrichment score (NES), P adj. ≤ 0.05 (FDR) and number of genes in the sample annotated per term (size) > 20. The running enrichment score is presented by a grey line for each of the ranked genes marked with a vertical black line. (H) The Reactome terms identified by the GSEA of the UP or DOWN genes in the variant shNEG versus mono, with NES ≥ 1.5 (absolute value), p value ≤ 0.05, and size > 30.

Article Snippet: Human chronic myeloid leukemia cell line K562 (#CCL-243) and bone marrow stroma fibroblast cell line HS-5 (#CRL-11882) were obtained from American Type Culture Collection (USA); human chronic myeloid leukemia cell line LAMA-84 (#ACC 168) was from DSMZ.

Techniques: Expressing, Luciferase, Injection, Variant Assay, Two Tailed Test, MANN-WHITNEY, Sequencing, Isolation, Fluorescence, FACS, Staining, Quantitative Proteomics

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days, when the GFP and human CD45 positive cells were FACS-sorted from xenografts for RNA isolation and sequencing. (B) Number of intersecting genes (compared groups indicated by black dot) that at the RNA level are upregulated (UP; log2 value of fold change ≥ 0.6) or downregulated (DOWN; log2 value of fold change ≤ -0.6) in shNEG cells xenografts imatinib versus vehicle treated and in CD34 positive cells from bone marrow biopsies of a patient with CML (data deposited at GEO under accession number GSE310243; from ) obtained after 6 months of imatinib therapy (combined samples SRR36072325 and SRR36072321) versus obtained at the diagnosis (combined SRR36072320 and SRR36072324). (B-D) Yellow shadow marks a group of genes that are UP or DOWN upon imatinib treatment in shNEG and CML cells (shNEG&CML), selected for subsequent analysis in (C) and (D). (C) Reactome terms identified by the GSEA analysis of the UP or DOWN genes in shNEG&CML with normalized enrichment score ≥ 1.5 (absolute value), p value ≤ 0.05, and number of genes in the sample annotated per term (size) ≥ 15. (C-D) The Reactome leading-edge genes identified by GSEA, annotated to terms marked with a brown triangle in (C) were selected to compare their expression level in single-cell RNA-seq data in (D) from bone marrow biopsies of healthy donors and CML patients with different responses to imatinib therapy. (D) Analysis of data at the Single-cell atlas of diagnostic Chronic Myeloid Leukemia bone marrow (scdbm) for CD34+ cells subtype . Data and detailed description of patient classification available: http://scdbm.ddnetbio.com ; A – responded to IM within 12 months; B – IM treatment failed within 18 months; C – resistant to IM and other TKI-s.

Techniques: Expressing, Luciferase, Injection, Isolation, Sequencing, Biomarker Discovery, Single Cell, RNA Sequencing, Diagnostic Assay

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Colony formation by K562 cells expressing shRNA non-targeting (shNEG) or targeting mRNA of TIAL1 (shTIAR), TIA1 (shTIA-1), or FMR1 (shFMRP) that were collected from ex vivo hypoxic (1.5% O2) co-culture with HS-5 bone marrow stromal fibroblasts and treated with 1 μM imatinib (IM) or 50nM Talazoparib (BMN) added in two doses following the experimental scheme (left panel). Number of colonies in each of the 3 technical replicates from 3-4 independent biological experiments presented as % change relative to the untreated cells (dashed black line) set as 100 %. Student’s two-tailed t-test was used to compare two samples marked by the black line; #### or **** - p<0.0001, ns - p > 0.05. (B) Scheme explaining the experimental setup based on the subcutaneous implantation in mice of 3D printed scaffolds (photo taken with a Samsung mobile phone camera) seeded with human cells differentiated into osteoblasts and K562/luc cells with shNEG, shTIAR or shTIA-1, followed by IM treatment for 14 days. (C) Bioluminescence signal monitored in mice after 2 weeks of IM or vehicle treatment (timeline explained in (B) ) following luciferin injection, collected in the Burker’s Xtreme In-Vivo chamber for 30 sec, and overlaid on the mouse X-ray image. Scale presents the signal intensity of the color coding. (D) Sum of the signal intensity (P) collected per second (s) and area (mm 2 ) for each mouse analyzed (single dot) is presented. (E) Scheme explaining experimental steps of xenograft formation by K562/luc (expressing shNEG or shTIAR, Firefly luciferase, and GFP) mixed with human primary bone marrow mesenchymal stem cells (hMSC) subcutaneously injected in mice 7 days before initiation of treatment with IM for the following 14 days. (F) Weight of each xenograft isolated from mice (single dot) formed by K562/luc cells with shNEG or shTIAR (as in (E) ) presented as fold change of the weight mean value of xenografts from mice treated with vehicle; mean value indicated with the black line. The nonparametric two-tailed Mann-Whitney test was used for comparisons indicated by black lines underneath the exact significance ( P ) values are presented in the plot. (A,D,F) Two-way Anova was used to compare shTIAR upon IM versus other variants; @ - P < 0.002.

Techniques: Expressing, shRNA, Ex Vivo, Co-Culture Assay, Two Tailed Test, Injection, In Vivo, Luciferase, Isolation, MANN-WHITNEY

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Comparison of gene expression in K562/luc cells with shNEG or shTIAR, FACS-sorted from xenografts (Xgraft), with level upon 2 weeks of IM versus vehicle (C) treatment changed with log2 value of fold change (Log2FC) ≥ 0.6 (UP) or ≤ -0.6 (DOWN); reversed regulation in shTIAR cells in purple. (B) Gene Ontology Molecular Function terms identified by the GSEA of genes that upon IM vs C in Xgraft of shNEG are reversely regulated in shTIAR (shaded in purple). In grey circles - UP (Log2FC > 0.6) n = 1993, brown circles DOWN (Log2FC < -0.6) n = 1756. Selected terms with NESabs ≥ 1.45, p-value ≤ 0.05, and the number of genes in the sample annotated per term (size) ≥ 10; n -number of genes. (C) Proportion of alternative splicing events (AS) changed upon shTIAR versus shNEG in cells from xenografts treated with vehicle (C) or imatinib (IM) for 2 weeks. Significant events from rMATS analysis requiring at least 20 reads/event, absolute change in PSI (Percent Spliced In) > 0.1 with FDR ≤ 0.05; total number in 3 experiments (n) above the bar. (D) Comparison of intron retention (RI; left panel) and cassette exon (CE; right panel) AS with significant PSI changed in shTIAR versus shNEG in cells from Xgraft or CO treated with IM. (E) Change in expression level of genes in Xgraft IM versus vehicle-treated for a subset of genes with significant changes shTIAR vs shNEG in RI (left panel) or SE (right panel) in cells from Xgraft and CO treated with IM; Log2FC ≤ -0.6 in brown, ≥ 0.6 in grey. (F) Difference in PSI value of CE, RI, and mutually exclusive exons (MXE) AS in: moro – shTIAR versus shNEG xenograft cells from mice treated with IM (Xgraft_IM); yellow – CD34 + enriched cells from bone marrow biopsies of a CML patient collected at 6-month IM therapy versus diagnosis GSE310243 (see in ). Included only with PSI > 0.1 and with FDR ≤ 0.05. (G) Expression level of genes with changes in AS (selected in (E) and (F) ) analyzed at the scdbm for the CD34 + cells subtype. Patient classification in .

Techniques: Comparison, Gene Expression, Alternative Splicing, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Scheme explaining experimental setup to determine proteomic changes using the quantitative BONCAT (QuaNCAT). K562 cells expressing shNEG or shTIAR growing in co-culture with HS-5 cells under hypoxia (1.5% O2) for 1.5 days were treated with imatinib (IM) for 18h before the bioorthogonal noncanonical amino acid tagging (BONCAT) of nascent proteins synthesized in cells for 4h. (B) Upper panel - number of genes and nascent proteins showing increased (UP; log₂ fold change [Log₂FC] ≥ 0.6) or decreased (DOWN; Log₂FC ≤ −0.6) abundance in IM-treated shTIAR cells compared with shNEG cells, with a significance threshold of p ≤ 0.05. In total, 2186 nascent proteins were quantified in the BONCAT experiment. Lower panel - scatter plot showing intensity-based absolute quantification (iBAQ), used as an estimate of relative protein molar abundance, plotted against Log₂FC values for BONCAT-identified proteins in shTIAR versus shNEG cells. Purple dots indicate hits with significant changes at both the protein and mRNA levels. Dashed vertical lines mark the Log₂FC thresholds of −0.6 and 0.6. (C) Functional annotation Gene Ontology Molecular Function enrichment analysis of UP (brown) or DOWN (purple) proteins with ClueGO/CytoScape, displaying proteins annotated with the term; only one side enriched terms with FDR < 0.05. (D) Lower panel - number of proteins that upon shTIAR are UP and DOWN regulated, for which mRNA was detected in the RNA immunoprecipitated (IP) in TIAR protein complexes (RIP), enriched in samples obtained with anti-TIAR antibody versus the same isotype non-binding antibody (ISO) (n=3647). Upper panel - example Image of Western blotting analysis of IP, with a sample of cell lysate used for IP loaded for reference (input). (E) Changes in the mRNA level, determined by real-time PCR and quantified using the ddCT method, expressed as log2 of change between IM-treated versus untreated cells, detected in samples from whole cells (upper panel) and anti-TIAR RIP (lower panel). Mean of 3 independent experiments with ± range is presented. Student’s t-test two-way was used to compare the difference between IM to C; * p ≤ 0.05, ** p≤0.005. (F) Number of genes identified in anti-TIAR RIP that show significant changes in intron retention (RI) or cassette exon alternative splicing (CE) in shTIAR versus shNEG alternative splicing analysis of RNA from IM-treated cells. Number of genes in the intersection provided above the bar; comparisons indicated by the black dot. (G) Sashimi plot (middle panel) demonstrating splicing of EIF4A2 mRNA within the region encompassing exons 8-11 and 3’UTR in RNA from anti-TIAR RIP. Alternative splice site usage marked by the purple line, and the percent usage ± ME (n=3) in numbers by the lines, reads coverage from 0-145 shown in grey, alternatively spliced exon marked by shaded yellow. Gene region scheme in the top. (H) Percent transcripts with TIAR-dependent alternative exon inclusion in K562 cells from xenografts treated with IM. Student’s t-test was used to compare results from three experiments (each dot) for the mean value marked with a thick black line; * p = 0.021.

Techniques: Expressing, Co-Culture Assay, Synthesized, Quantitative Proteomics, Functional Assay, Immunoprecipitation, Binding Assay, Western Blot, Real-time Polymerase Chain Reaction, Alternative Splicing

Journal: bioRxiv

Article Title: TIAR-dependent coordination of alternative splicing and lipid peroxidation is required for CML cell resistance to imatinib in the bone marrow stroma

doi: 10.64898/2026.05.29.728710

Figure Lengend Snippet: (A) Expression level of genes analyzed at the scdbm for CD34 + cells subtype; patient classification explained in . (B-H) Impact of shTIAR compared to shNEG analyzed in K562 cells that were co-cultured with HS-5 cells under hypoxia (1.5%O2) 2 days before initiation of 18h culture with imatinib (IM) treatment or without (C). (B) Protein level in whole cell extracts analyzed by Western blot, representative images of immunoblots (n=3) presented. (C) Mitochondrial (mit.) membrane potential measured using JC-1 probe; values for signal from probe aggregates (red) in polarized mitochondria expressed as % of signal from the probe in the cells (n=3), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) used as a control. (C,D,F,H) Bars represent the mean of values from independent experiments (indicated by dots) with ± SD. Student’s t-test was used to determine the significance of the difference shTIAR vs shNEG (#) and IM vs C (*); * - p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.005, **** p ≤ 0.001 (D) Lipid peroxidation measured with click-it chemistry by flow cytometry. Fluorescence intensity GeoMean expressed as fold change of value in the untreated shNEG cells (n=5). (E-H) Presentation on the cluster of cell differentiation (CD) surface protein markers CD45 and CD235a on K562 cells with shNEG or shTIAR isolated from co-culture established in hypoxia (1.5%O2) a day before initiation of treatment with IM for 48h (E-G) or FACS-sorted K562/luc GFP positive cells from xenografts (H) . (E-F,H) Percentage of parental live cell subpopulations that are: double positive for CD45 and CD235a (CD235a&CD45), positive only for CD45, or only for CD235a, or negative for both CD markers. Representative scatter plots in (E) , summary of independent co-culture experiments (n=4) in (F) . (G) Fluorescence intensity of surface CD36 protein staining in the fraction of cells positive for CD235a or CD45. Numbers correspond to fold change in shTIAR to shNEG ± ME (n=2). (H) Percent GFP and hCD45-positive cells from different xenografts (n=4) that are positive for CD235a. Two-way ANOVA test was used to for comparison of shNEG_C to other variants (@), and p=0.0046.

Techniques: Expressing, Cell Culture, Western Blot, Membrane, Control, Flow Cytometry, Fluorescence, Cell Differentiation, Isolation, Co-Culture Assay, Staining, Comparison

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).

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

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

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

The Effect of lovastatin on the activity, cell cycle, and apoptosis in AML cell lines K562 and THP-1 in vitro. ( A and B ) Effects of lovastatin and 4-PBA at different concentrations on cell activities of THP-1 and K562; ( C ) Effects of lovastatin and 4-PBA on cell cycles of THP-1 and K562; ( D ) Effects of lovastatin and 4-PBA on cell apoptosis of THP-1 and K562. AML, acute myeloid leukemia. Cells were treated with lovastatin (100 μM) or 4-PBA (5 μM) for 24 hours; **** p <0.0001.

Journal: International Journal of General Medicine

Article Title: Lovastatin Targets LIPA to Induce ER Stress-Mediated Apoptosis in Acute Myeloid Leukemia: A Multi-Omics Study

doi: 10.2147/IJGM.S591023

Figure Lengend Snippet: The Effect of lovastatin on the activity, cell cycle, and apoptosis in AML cell lines K562 and THP-1 in vitro. ( A and B ) Effects of lovastatin and 4-PBA at different concentrations on cell activities of THP-1 and K562; ( C ) Effects of lovastatin and 4-PBA on cell cycles of THP-1 and K562; ( D ) Effects of lovastatin and 4-PBA on cell apoptosis of THP-1 and K562. AML, acute myeloid leukemia. Cells were treated with lovastatin (100 μM) or 4-PBA (5 μM) for 24 hours; **** p <0.0001.

Article Snippet: The human leukemia cell lines K562 (ATCC ® CCL-243TM) and THP-1 (ATCC ® TIB-202TM) were commercially obtained from Sangon Biotech (Shanghai, China).

Techniques: Activity Assay, In Vitro

The mRNA expression levels of LIPA, ATF6, eIF2α, XBP1, IRE1A, DDIT3, HSP90AA1, and PERK. ( A-H ) The ER biomarkers expression in K562 cells ( I-P ). The ER biomarkers expression in THP-1 cells. Cells were treated with lovastatin (100 μM) or 4-PBA (5 μM) for 24 h; **** p <0.0001.

Journal: International Journal of General Medicine

Article Title: Lovastatin Targets LIPA to Induce ER Stress-Mediated Apoptosis in Acute Myeloid Leukemia: A Multi-Omics Study

doi: 10.2147/IJGM.S591023

Figure Lengend Snippet: The mRNA expression levels of LIPA, ATF6, eIF2α, XBP1, IRE1A, DDIT3, HSP90AA1, and PERK. ( A-H ) The ER biomarkers expression in K562 cells ( I-P ). The ER biomarkers expression in THP-1 cells. Cells were treated with lovastatin (100 μM) or 4-PBA (5 μM) for 24 h; **** p <0.0001.

Article Snippet: The human leukemia cell lines K562 (ATCC ® CCL-243TM) and THP-1 (ATCC ® TIB-202TM) were commercially obtained from Sangon Biotech (Shanghai, China).

Techniques: Expressing

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.

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

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human chronic myeloid leukemia cell line k562 (ATCC)

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