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DSMZ cell culture 32d
Cell Culture 32d, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cell culture 32d - by Bioz Stars, 2026-05

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Validation of a Btk site-specific gene editing approach (A) Schematic of Btk editing strategy. The top row shows the Btk gene with exons and introns. In the second row, zooming in on intron 1 and exon 2 of the Btk gene, shows the sites where sgRNAs targeting these regions will allow for Cas9 dsDNA breaks. In the third row, recombinant adeno-associated virus (rAAV) vectors containing a nearly full-length human BTK cDNA with 500-bp homology arms directly flanking the dsDNA break site to allow for homologous recombination at either the intron 1 or exon 2 sites. The BTK cDNA donor also contains a “micro” version of the terminal intron 18, Btk 3′ UTR, and WPRE element. The bottom row depicts the BTK minigene inserted into the endogenous murine Btk locus at the intron 1 or exon 2 sites. (B) Outline displaying 32D and Lin- cell editing timeline. Cells are cultured in either R-10 or SFEM + cytokines for the times listed, then electroporated with Cas9 RNP followed directly by transduction with rAAV6 donor. One week post editing, genomic DNA and total protein lysates are harvested for analysis of allelic disruption, gene integration, and exogenous Btk protein expression. (C and G) Genomic DNA harvested from 32D and Lin- cells, respectively, was PCR amplified with primers flanking the Cas9/sgRNA cut sites and sent for Sanger sequencing analysis of Btk allelic disruption via synthego ICE analysis. The y axis represents the frequency of amplified DNA copies which contained insertions/deletions at the Btk dsDNA break site. n = 3. (D and H) Twenty-four hours post electroporation, 32D and Lin- cells, respectively, were analyzed for viable cell counts using hemacytometers and trypan blue exclusion to determine the acute toxic effects of editing reagents. The y axis represents the average percentage of live cells counted across two individual aliquots of a given sample. (E and I) Genomic DNA harvested from 32D and Lin- cells, respectively, underwent in/out ddPCR analysis using primers and probes that allow for specific quantification of BTK cDNA insertion at its endogenous locus to quantify Btk site-specific integration frequency. The y axis represents the frequency of site-specific Btk integration events normalized to a reference housekeeping gene. n = 3. (F and J) Western blot protein analysis of Btk expression from Btk −/− 32D cells and Btk/Tec −/− Lin- cells, respectively. Cells were lysed using RIPA lysis buffer followed by western blotting and chemiluminescent detection of Btk and Beta-Actin protein expression levels; 32D cells were transfected with equivalent Cas9-RNPs followed by transduction of rAAV6 at MOIs of either 1e5 or 5e5. To determine the role of rAAV6 transduction alone on exogenous Btk expression, Lin- cells either received Cas9-RNP or no electroporation, followed by transduction with rAAV6 at an MOI of 5e5. n = 3.

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Hematopoietic stem cell gene therapy for the treatment of X-linked agammaglobulinemia

doi: 10.1016/j.omtm.2025.101555

Figure Lengend Snippet: Validation of a Btk site-specific gene editing approach (A) Schematic of Btk editing strategy. The top row shows the Btk gene with exons and introns. In the second row, zooming in on intron 1 and exon 2 of the Btk gene, shows the sites where sgRNAs targeting these regions will allow for Cas9 dsDNA breaks. In the third row, recombinant adeno-associated virus (rAAV) vectors containing a nearly full-length human BTK cDNA with 500-bp homology arms directly flanking the dsDNA break site to allow for homologous recombination at either the intron 1 or exon 2 sites. The BTK cDNA donor also contains a “micro” version of the terminal intron 18, Btk 3′ UTR, and WPRE element. The bottom row depicts the BTK minigene inserted into the endogenous murine Btk locus at the intron 1 or exon 2 sites. (B) Outline displaying 32D and Lin- cell editing timeline. Cells are cultured in either R-10 or SFEM + cytokines for the times listed, then electroporated with Cas9 RNP followed directly by transduction with rAAV6 donor. One week post editing, genomic DNA and total protein lysates are harvested for analysis of allelic disruption, gene integration, and exogenous Btk protein expression. (C and G) Genomic DNA harvested from 32D and Lin- cells, respectively, was PCR amplified with primers flanking the Cas9/sgRNA cut sites and sent for Sanger sequencing analysis of Btk allelic disruption via synthego ICE analysis. The y axis represents the frequency of amplified DNA copies which contained insertions/deletions at the Btk dsDNA break site. n = 3. (D and H) Twenty-four hours post electroporation, 32D and Lin- cells, respectively, were analyzed for viable cell counts using hemacytometers and trypan blue exclusion to determine the acute toxic effects of editing reagents. The y axis represents the average percentage of live cells counted across two individual aliquots of a given sample. (E and I) Genomic DNA harvested from 32D and Lin- cells, respectively, underwent in/out ddPCR analysis using primers and probes that allow for specific quantification of BTK cDNA insertion at its endogenous locus to quantify Btk site-specific integration frequency. The y axis represents the frequency of site-specific Btk integration events normalized to a reference housekeeping gene. n = 3. (F and J) Western blot protein analysis of Btk expression from Btk −/− 32D cells and Btk/Tec −/− Lin- cells, respectively. Cells were lysed using RIPA lysis buffer followed by western blotting and chemiluminescent detection of Btk and Beta-Actin protein expression levels; 32D cells were transfected with equivalent Cas9-RNPs followed by transduction of rAAV6 at MOIs of either 1e5 or 5e5. To determine the role of rAAV6 transduction alone on exogenous Btk expression, Lin- cells either received Cas9-RNP or no electroporation, followed by transduction with rAAV6 at an MOI of 5e5. n = 3.

Article Snippet: 32D cells (ATCC CRL-11346) were electroporated at 85% confluency.

Techniques: Biomarker Discovery, Recombinant, Virus, Homologous Recombination, Cell Culture, Transduction, Disruption, Expressing, Amplification, Sequencing, Electroporation, Western Blot, Lysis, Transfection

OSM is induced by STAT5-activating oncogenes in a kinase-dependent manner and acts on bone marrow stromal cells via STAT3 phosphorylation. a 32D cells were transduced with various oncogenes and starved of cytokines and serum for twelve hours. Quantitative PCR revealed Osm upregulation in BCR::ABL1-, FLT3-ITD-, NPM1::ALK-, and JAK2 p.V617F-positive cells. Osm expression values were normalized to Gapdh and empty vector (MiG-empty) expression values. N ≥ 3 experiments; p values (compared with MiG-empty cells): *** p < 0.001; ** p < 0.01. The data are presented as the means ± SEMs. b 32D cells transduced with oncogenes were serum-starved plus inhibitor-treated as indicated for twelve hours. Osm expression values were normalized to Gapdh and MiG-empty vector expression values. P values (compared with MiG-empty cells): *** p < 0.001; ** p < 0.01. The data are presented as the means ± SEMs. c Flow cytometric analysis of OSMR surface levels in primary human stromal cells, primary AML blasts, and human myeloid leukemic cell lines. The mean fluorescence intensity (MFI) of the isotype control was subtracted from the OSMR MFI values. Each dot represents one measurement. The data are presented as the means ± SEMs. d Primary murine hematopoietic BM cells (HCs) did not express Osmr transcripts after 40 PCR cycles, whereas mesenchymal stromal cell (MSC) populations expressed Osmr . MSC = mesenchymal stromal cells, OB = osteoblasts, ND = not detected. Each dot represents one measurement. The data are presented as the means ± SEMs. e Flow cytometric analysis of pSTAT3 levels in primary human stromal cells and human myeloid leukemic cell lines after huOSM treatment (2.5 h, 10 ng/mL). The mean fluorescence intensity (MFI) of the isotype control was subtracted from the pSTAT3 antibody MFI values. Each dot represents one measurement. The data are presented as the means ± SEMs. f , g OSM ( f ) and OSMR ( g ) expression across hematological and solid neoplasms utilizing Expression Public 24Q2 and lineage subtype grouping on the DepMap portal ( https://depmap.org )

Journal: Signal Transduction and Targeted Therapy

Article Title: Oncostatin M induced by STAT5-activating oncogenes promotes disease progression in hematologic malignancies

doi: 10.1038/s41392-025-02491-6

Figure Lengend Snippet: OSM is induced by STAT5-activating oncogenes in a kinase-dependent manner and acts on bone marrow stromal cells via STAT3 phosphorylation. a 32D cells were transduced with various oncogenes and starved of cytokines and serum for twelve hours. Quantitative PCR revealed Osm upregulation in BCR::ABL1-, FLT3-ITD-, NPM1::ALK-, and JAK2 p.V617F-positive cells. Osm expression values were normalized to Gapdh and empty vector (MiG-empty) expression values. N ≥ 3 experiments; p values (compared with MiG-empty cells): *** p < 0.001; ** p < 0.01. The data are presented as the means ± SEMs. b 32D cells transduced with oncogenes were serum-starved plus inhibitor-treated as indicated for twelve hours. Osm expression values were normalized to Gapdh and MiG-empty vector expression values. P values (compared with MiG-empty cells): *** p < 0.001; ** p < 0.01. The data are presented as the means ± SEMs. c Flow cytometric analysis of OSMR surface levels in primary human stromal cells, primary AML blasts, and human myeloid leukemic cell lines. The mean fluorescence intensity (MFI) of the isotype control was subtracted from the OSMR MFI values. Each dot represents one measurement. The data are presented as the means ± SEMs. d Primary murine hematopoietic BM cells (HCs) did not express Osmr transcripts after 40 PCR cycles, whereas mesenchymal stromal cell (MSC) populations expressed Osmr . MSC = mesenchymal stromal cells, OB = osteoblasts, ND = not detected. Each dot represents one measurement. The data are presented as the means ± SEMs. e Flow cytometric analysis of pSTAT3 levels in primary human stromal cells and human myeloid leukemic cell lines after huOSM treatment (2.5 h, 10 ng/mL). The mean fluorescence intensity (MFI) of the isotype control was subtracted from the pSTAT3 antibody MFI values. Each dot represents one measurement. The data are presented as the means ± SEMs. f , g OSM ( f ) and OSMR ( g ) expression across hematological and solid neoplasms utilizing Expression Public 24Q2 and lineage subtype grouping on the DepMap portal ( https://depmap.org )

Article Snippet: 32D, NIH/3T3 and OP9 cells were obtained from the German Resource Centre for Biological Material (DSMZ).

Techniques: Phospho-proteomics, Transduction, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Fluorescence, Control

a IL-1β, IL-18, TNFα and HMGB1 serum concentrations in Jak2 VF , WT, Jak2 VF ;Nlrp3 −/− and Nlrp3 −/− mice (n = 9 mice/group). Dots represent individual mice. b BMDMs from Jak2 VF and WT mice were stimulated or left untreated as indicated, and cell lysates and supernatants were assessed for pro-IL-1ß and cleaved IL-1ß by immunoblotting. The blot is representative of 2 independent experiments. c Caspase 1 activation in 32D cells expressing JAK2V617F, CALRdel52, CALRins5 or an empty vector (EV, no oncogene). For 32D EV cells, the medium was supplemented with murine IL-3. Each dot represents an independent experiment using separately cultured cell populations (n = 3 per group). d Representative images of bone marrow sections from Jak2 VF , WT and Nlrp3 −/− mice at 52 weeks of age stained for NLRP3. Scale bar equals 100 µm. e NLRP3 staining intensity score of bone marrow sections from Jak2 VF and WT mice. Dots represent individual mice and horizontal lines the median (n = 4 mice/group). Scatter bar plots in ( a , c ) show mean + SEM. Statistically significant differences were determined by one-way ANOVA with two-sided Holm-Šidák multiple comparison test ( a ) or one-way ANOVA with two-sided Dunnett's multiple comparison test ( c ), or by two-tailed unpaired Mann-Whitney U test ( e ). Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: NLRP3-induced systemic inflammation controls the development of JAK2V617F mutant myeloproliferative neoplasms

doi: 10.1038/s41467-025-65673-4

Figure Lengend Snippet: a IL-1β, IL-18, TNFα and HMGB1 serum concentrations in Jak2 VF , WT, Jak2 VF ;Nlrp3 −/− and Nlrp3 −/− mice (n = 9 mice/group). Dots represent individual mice. b BMDMs from Jak2 VF and WT mice were stimulated or left untreated as indicated, and cell lysates and supernatants were assessed for pro-IL-1ß and cleaved IL-1ß by immunoblotting. The blot is representative of 2 independent experiments. c Caspase 1 activation in 32D cells expressing JAK2V617F, CALRdel52, CALRins5 or an empty vector (EV, no oncogene). For 32D EV cells, the medium was supplemented with murine IL-3. Each dot represents an independent experiment using separately cultured cell populations (n = 3 per group). d Representative images of bone marrow sections from Jak2 VF , WT and Nlrp3 −/− mice at 52 weeks of age stained for NLRP3. Scale bar equals 100 µm. e NLRP3 staining intensity score of bone marrow sections from Jak2 VF and WT mice. Dots represent individual mice and horizontal lines the median (n = 4 mice/group). Scatter bar plots in ( a , c ) show mean + SEM. Statistically significant differences were determined by one-way ANOVA with two-sided Holm-Šidák multiple comparison test ( a ) or one-way ANOVA with two-sided Dunnett's multiple comparison test ( c ), or by two-tailed unpaired Mann-Whitney U test ( e ). Source data are provided as a Source Data file.

Article Snippet: 32D cells (DSMZ, ACC 411) transduced with the pMSCV-MPL-HA-IRES-puromycin vector followed by a second transduction with the empty pMSCV-IRES-GFP vector or pMSCV-IRES-GFP containing the JAK2V617F, CALRdel52 or CALRins5 oncogene were described previously .

Techniques: Western Blot, Activation Assay, Expressing, Plasmid Preparation, Cell Culture, Staining, Comparison, Two Tailed Test, MANN-WHITNEY

(a) Efficient shRNA-mediated knockdown of HMGA1 (sh1, sh2 vs. sh-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; ACTB served as loading control. (b) Lentiviral-mediated overexpression of HMGA1 (OE vs. CMV-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; Tubulin served as loading control. (c) Lentiviral-mediated overexpression of Hmga1 (J/OE vs. J/NC control) in murine Ba/F3 ( Jak2 wild type, or Jak2 V617F ) and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells. Left: Relative Hmga1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of Hmga1 protein; Tubulin served as loading control. Statistical analyses for (a-c) by two-sample t-test or one-way ANOVA, as appropriate. (d) HMGA1 overexpression exacerbates disease phenotype in a HEL xenograft model. Hematological parameters (WBC, white blood cell count; HGB, hemoglobin; HCT, hematocrit; PLT, platelet count) in NSG mice engrafted with HEL cells stably expressing control vector (CMV-NC, n = 6) or HMGA1 (OE, n = 6) at 35 days post-transplantation. Data are presented as mean ± SD. Two-sample t -test. (e) HMGA1 knockdown alters chromatin accessibility and HMGA1 binding at key cell cycle regulatory gene loci. Integrative Genomics Viewer (IGV) snapshots displaying ATAC-seq and HMGA1 CUT&Tag signals at representative E2F target genes ( E2F1 , CCNE1 , CCNE2 , CDK2 , RB1 ), G2M checkpoint genes ( CCNB1 , CCNB2 , CDC2 , WEE1 , CDC25C , PLK1 , AURKA , AURKB ), and common cell cycle regulators ( CCNA2 , CDKN1A / p21 , CDKN1B / p27 ) in HEL cells following control (NC) versus HMGA1 knockdown (KD). (f) Enhanced E2F target and G2M checkpoint gene signatures in sAML patient cells. UMAP projections of single-cell CITE-seq data (GSE185381) from control and sAML patients, with cells colored by enrichment scores for E2F target and G2M checkpoint gene sets. Corresponding density plots illustrate score distributions.

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Efficient shRNA-mediated knockdown of HMGA1 (sh1, sh2 vs. sh-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; ACTB served as loading control. (b) Lentiviral-mediated overexpression of HMGA1 (OE vs. CMV-NC control) in HEL and UKE-1 cells. Left: Relative HMGA1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of HMGA1 protein; Tubulin served as loading control. (c) Lentiviral-mediated overexpression of Hmga1 (J/OE vs. J/NC control) in murine Ba/F3 ( Jak2 wild type, or Jak2 V617F ) and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells. Left: Relative Hmga1 mRNA levels by qRT-PCR (mean ± SD, n = 3). Right: Western blot analysis of Hmga1 protein; Tubulin served as loading control. Statistical analyses for (a-c) by two-sample t-test or one-way ANOVA, as appropriate. (d) HMGA1 overexpression exacerbates disease phenotype in a HEL xenograft model. Hematological parameters (WBC, white blood cell count; HGB, hemoglobin; HCT, hematocrit; PLT, platelet count) in NSG mice engrafted with HEL cells stably expressing control vector (CMV-NC, n = 6) or HMGA1 (OE, n = 6) at 35 days post-transplantation. Data are presented as mean ± SD. Two-sample t -test. (e) HMGA1 knockdown alters chromatin accessibility and HMGA1 binding at key cell cycle regulatory gene loci. Integrative Genomics Viewer (IGV) snapshots displaying ATAC-seq and HMGA1 CUT&Tag signals at representative E2F target genes ( E2F1 , CCNE1 , CCNE2 , CDK2 , RB1 ), G2M checkpoint genes ( CCNB1 , CCNB2 , CDC2 , WEE1 , CDC25C , PLK1 , AURKA , AURKB ), and common cell cycle regulators ( CCNA2 , CDKN1A / p21 , CDKN1B / p27 ) in HEL cells following control (NC) versus HMGA1 knockdown (KD). (f) Enhanced E2F target and G2M checkpoint gene signatures in sAML patient cells. UMAP projections of single-cell CITE-seq data (GSE185381) from control and sAML patients, with cells colored by enrichment scores for E2F target and G2M checkpoint gene sets. Corresponding density plots illustrate score distributions.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: shRNA, Knockdown, Control, Quantitative RT-PCR, Western Blot, Over Expression, Cell Counting, Stable Transfection, Expressing, Plasmid Preparation, Transplantation Assay, Binding Assay

(a) Relative proliferation curves of human (HEL, UKE-1) and murine (Ba/F3, 32D-cl3 transduced with Jak2 wild-type or Jak2 V617F ) cell lines following HMGA1/Hmga1 overexpression (OE) or shRNA-meidated knockdown (sh1, sh2) compared to respective controls (CMV-NC or sh-NC)NC.) 32D-cl3 cells were cultured with IL-3. Data are mean ± SD. (n = 5 per group). Two-way ANOVA. (b) Flow cytometric analysis of CD11b expression on 32D-cl3 cells transduced with Jak2 wild-type (J WT ) or Jak2 V617F (J VF ), and co-transduced with control vector (NC) or HMGA1 overexpression (OE), following G-CSF (100 ng/mL) induced differentiation. (i) Representative histograms of CD11b-FITC fluorescence. (ii) Quantification of HMGA1-PE mean fluorescence intensity (MFI). (iii) Quantification of CD11b-FITC MFI (n = 5 per group). Data are mean ± SD. Two-sample t -test. (c) Quantification of human CD45 + CD117 + HEL cells in peripheral blood of NSG mice at day 35 post-transplant, comparing HMGA1-OE versus vector control (CMV-NC) groups (n=6 per group). Data are mean ± SD. Two-sample t -test. (d) Wright-Giemsa stained peripheral blood smears from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells at day 35. Quantification of HEL cells (% of total nucleated cells) is shown (n = 6 per group). Data are mean ± SD. Two-sample t -test. (e-f) Representative H&E and HMGA1 IHC staining (left panels of e and f, respectively) and quantification of HMGA1-positive cells (%) (right panels fo e and f, respectively) in (e) femur bone marrow and (f) spleen sections from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells. Scale bars: 50 µm. Data are mean ± SD. Two-sample t -test. (g) Representative images of spleens (left) and relative spleen weights (spleen weight/body weight %, right) from NSG mice at day 35 post-engraftment with HMGA1-OE or CMV-NC HEL cells (n = 6 per group). Data are mean ± SD. Two-sample t -test. (h) Kaplan-Meier survival curves for NSG mice injected with HMGA1-OE ro CMV-NC HEL cells (n = 6 per group). Median survival times are indicated. Log-rank (Mantel-Cox) test. (i) Heatmaps showing HMGA1 binding intensity (CUT&Tag, left) and chromatin accessibility (ATAC-seq, right) centered on transcription start site (TSS ± 3kb) for genes in HEL cells transduced with shNC or shHMGA1. Color scale indicates normalized read counts (Max/Min normalized). (j) Top de novo motifs identified by HOMER analysis within ATAC-seq peak regions that either lose accessibility (left) or gain accessibility (right) upon HMGA1 knockdown in HEL cells. P -value for motif enrichment are indicated. (k) Quantification of apoptosis by Annexin V-APC/7-AAD staining and flow cytometry in HEL and UKE-1 cells after transduction with shNC or HMGA1 shRNAs (sh1, sh2). Representative flow cytometry plots are shown. Data are mean ± SD. (n = 5 per group). One-way ANOVA with Tukey’s post-hoc test.

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a) Relative proliferation curves of human (HEL, UKE-1) and murine (Ba/F3, 32D-cl3 transduced with Jak2 wild-type or Jak2 V617F ) cell lines following HMGA1/Hmga1 overexpression (OE) or shRNA-meidated knockdown (sh1, sh2) compared to respective controls (CMV-NC or sh-NC)NC.) 32D-cl3 cells were cultured with IL-3. Data are mean ± SD. (n = 5 per group). Two-way ANOVA. (b) Flow cytometric analysis of CD11b expression on 32D-cl3 cells transduced with Jak2 wild-type (J WT ) or Jak2 V617F (J VF ), and co-transduced with control vector (NC) or HMGA1 overexpression (OE), following G-CSF (100 ng/mL) induced differentiation. (i) Representative histograms of CD11b-FITC fluorescence. (ii) Quantification of HMGA1-PE mean fluorescence intensity (MFI). (iii) Quantification of CD11b-FITC MFI (n = 5 per group). Data are mean ± SD. Two-sample t -test. (c) Quantification of human CD45 + CD117 + HEL cells in peripheral blood of NSG mice at day 35 post-transplant, comparing HMGA1-OE versus vector control (CMV-NC) groups (n=6 per group). Data are mean ± SD. Two-sample t -test. (d) Wright-Giemsa stained peripheral blood smears from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells at day 35. Quantification of HEL cells (% of total nucleated cells) is shown (n = 6 per group). Data are mean ± SD. Two-sample t -test. (e-f) Representative H&E and HMGA1 IHC staining (left panels of e and f, respectively) and quantification of HMGA1-positive cells (%) (right panels fo e and f, respectively) in (e) femur bone marrow and (f) spleen sections from NSG mice engrafted with HMGA1-OE or CMV-NC HEL cells. Scale bars: 50 µm. Data are mean ± SD. Two-sample t -test. (g) Representative images of spleens (left) and relative spleen weights (spleen weight/body weight %, right) from NSG mice at day 35 post-engraftment with HMGA1-OE or CMV-NC HEL cells (n = 6 per group). Data are mean ± SD. Two-sample t -test. (h) Kaplan-Meier survival curves for NSG mice injected with HMGA1-OE ro CMV-NC HEL cells (n = 6 per group). Median survival times are indicated. Log-rank (Mantel-Cox) test. (i) Heatmaps showing HMGA1 binding intensity (CUT&Tag, left) and chromatin accessibility (ATAC-seq, right) centered on transcription start site (TSS ± 3kb) for genes in HEL cells transduced with shNC or shHMGA1. Color scale indicates normalized read counts (Max/Min normalized). (j) Top de novo motifs identified by HOMER analysis within ATAC-seq peak regions that either lose accessibility (left) or gain accessibility (right) upon HMGA1 knockdown in HEL cells. P -value for motif enrichment are indicated. (k) Quantification of apoptosis by Annexin V-APC/7-AAD staining and flow cytometry in HEL and UKE-1 cells after transduction with shNC or HMGA1 shRNAs (sh1, sh2). Representative flow cytometry plots are shown. Data are mean ± SD. (n = 5 per group). One-way ANOVA with Tukey’s post-hoc test.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: Transduction, Over Expression, shRNA, Knockdown, Cell Culture, Expressing, Control, Plasmid Preparation, Fluorescence, Staining, Immunohistochemistry, Injection, Binding Assay, Flow Cytometry

(a-f) HMGA1 expression levels modulate sensitivity to diverse therapeutic agents. Dose-response curves showing viability of HEL, UKE-1, Ba/F3 ( Jak2 wild type, or Jak2 V617F ), and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells with engineered HMGA1/Hmga1 expression (OE vs. NC; sh1/sh2 vs. sh-NC) following 72-hour treatment with (a) IFNα, (b) 5-Azacytidine, (c) Decitabine, (d) Cytarabine, (e) Venetoclax, and (f) Hydroxyurea. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (g) Ruxolitinib treatment, particularly long-term exposure, alters key signaling and cell cycle protein expression. Western blot analysis of indicated JAK-STAT, E2F pathway, G2M checkpoint, and cell cycle regulatory proteins in HEL and UKE-1 cells treated with vehicle, short-term ruxolitinib (4 hours), or in ruxolitinib-persistent (Rux-P) lines. GAPDH served as loading control. (h-j) HMGA1/Hmga1 expression status influences sensitivity to JAK inhibitors. Dose-response curves assessing viability of (h) UKE-1 cells, (i) Ba/F3 cells (J VF /NC: Jak2 V617F /control vector; J VF /OE: Jak2 V617F /Hmga1 OE; J WT /NC: Jak2 wild-type/control vector; J WT /OE: Jak2 wild-type /Hmga1 OE), and (j) 32D-cl3 cells (similarly engineered) with engineered HMGA1/Hmga1 expression, following treatment with ruxolitinib, fedratinib, pacritinib, or momelotinib. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (k) Pacritinib mitigates weight loss in mice bearing HMGA1-overexpressing HEL xenografts. Percent body weight change in NSG mice engrafted with HEL-Luc cells (CMV-NC or HMGA1-OE) and treated with vehicle or pacritinib (100 mg/kg, BID, 14 days). Data are mean ± SD (n = 6 per group). One-way ANOVA. (l) Pacritinib treatment improves hematological parameters in the HMGA1-overexpressing HEL xenograft model. Peripheral blood counts (WBC, HGB, HCT, PLT) in xenografted mice at day 35 endpoint. Data are mean ± SD (n = 6 per group). One-way ANOVA.

Journal: bioRxiv

Article Title: Targeting HMGA1-driven leukemic transformation in myeloproliferative neoplasms with pacritinib

doi: 10.1101/2025.06.01.657170

Figure Lengend Snippet: (a-f) HMGA1 expression levels modulate sensitivity to diverse therapeutic agents. Dose-response curves showing viability of HEL, UKE-1, Ba/F3 ( Jak2 wild type, or Jak2 V617F ), and 32D-cl3 ( Jak2 wild type, or Jak2 V617F ) cells with engineered HMGA1/Hmga1 expression (OE vs. NC; sh1/sh2 vs. sh-NC) following 72-hour treatment with (a) IFNα, (b) 5-Azacytidine, (c) Decitabine, (d) Cytarabine, (e) Venetoclax, and (f) Hydroxyurea. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (g) Ruxolitinib treatment, particularly long-term exposure, alters key signaling and cell cycle protein expression. Western blot analysis of indicated JAK-STAT, E2F pathway, G2M checkpoint, and cell cycle regulatory proteins in HEL and UKE-1 cells treated with vehicle, short-term ruxolitinib (4 hours), or in ruxolitinib-persistent (Rux-P) lines. GAPDH served as loading control. (h-j) HMGA1/Hmga1 expression status influences sensitivity to JAK inhibitors. Dose-response curves assessing viability of (h) UKE-1 cells, (i) Ba/F3 cells (J VF /NC: Jak2 V617F /control vector; J VF /OE: Jak2 V617F /Hmga1 OE; J WT /NC: Jak2 wild-type/control vector; J WT /OE: Jak2 wild-type /Hmga1 OE), and (j) 32D-cl3 cells (similarly engineered) with engineered HMGA1/Hmga1 expression, following treatment with ruxolitinib, fedratinib, pacritinib, or momelotinib. Calculated IC50 values are shown. Data represent mean ± SD from n = 3 independent experiments. Two-way ANOVA. (k) Pacritinib mitigates weight loss in mice bearing HMGA1-overexpressing HEL xenografts. Percent body weight change in NSG mice engrafted with HEL-Luc cells (CMV-NC or HMGA1-OE) and treated with vehicle or pacritinib (100 mg/kg, BID, 14 days). Data are mean ± SD (n = 6 per group). One-way ANOVA. (l) Pacritinib treatment improves hematological parameters in the HMGA1-overexpressing HEL xenograft model. Peripheral blood counts (WBC, HGB, HCT, PLT) in xenografted mice at day 35 endpoint. Data are mean ± SD (n = 6 per group). One-way ANOVA.

Article Snippet: Murine IL-3-dependent pro-B cell line Ba/F3 (DSMZ ACC 300) and myeloblastic cell line 32D-cl3 (ATCC CRL-11346) were also used.

Techniques: Expressing, Western Blot, Control, Plasmid Preparation

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