cell culture 32d  (DSMZ)

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

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

Journal: bioRxiv

Article Title: Leukemic Cells Manipulate MSCs Bioelectrical Signals to Reshape the Bone Marrow Niche

doi: 10.1101/2025.03.10.642319

Figure Lengend Snippet: (A) Cell proliferation of AML-MSCs, h-MSCs, and h-MSCs treated with 40 mM K + gluconate or 10 nM Ouabain by Presto Blue assay (n=5). Data were normalized to time 0-hour samples (t-test comparing all groups versus h-MSCs). (B-C) Cell density of murine IL-3–dependent 32D cell line cultured in the presence (black bar) or absence of IL-3 (blue bar), compared with 32D cell line cultured on a layer of AML-MSCs (red bar), h-MSCs (grey) or h-MSCs pre-treated for 72 hours with Ouabain (B, n=5) or with K + gluconate (C, n=5). T-test was performed to compare treated groups or AML-MSCs versus h-MSCs, with ±IL3 used as experimental control. (D) Total branches length of HUVEC tubes by using conditioned medium derived from AML-MSCs (n=7) and h-MSCs pre-treated or not for 72 hours with Ouabain or K + gluconate and then stimulated (st) or not (unst) with a pro-inflammatory cytokine cocktail (hIL-1β, hIL-6, and hTNF-α) for 24 hours; tube formation was evaluated after 4 hours and normalized to unst condition (AU: arbitrary unit, n=4, t-test comparing treated or AML-MSCs stimulated groups versus stimulated h-MSCs). (E) Percentage of PHA-stimulated CD3 + T cells expressing CD69 and CD25 after 72 hours of co-culture with AML-MSCs and h-MSCs pre-treated for 72 hours with Ouabain or K + gluconate, relative to SF condition (without MSCs) (n=4, t-test comparing treated or AML-MSCs groups versus h-MSCs). (F) Relative expression measured by RQ-PCR of IL-6 (interleukin-6) in h-MSCs pre-treated or not for 72 hours with K + gluconate (n=4) and in AML-MSCs (n=3, t-test comparing treated or AML-MSCs groups versus h-MSCs). (G) IL[6 protein secretion levels (pg/mL), measured by ELISA, in AML-MSCs (n=24) or in h-MSCs pre-treated or not for 72 hours with K + gluconate (n=4 or n=6 respectively), relative to h-MSCs untreated condition (t-test comparing treated or AML-MSCs groups versus h-MSCs). (H-J) Relative RQ-PCR expression of osteoprogenitor-associated genes TNAP (Tissue-nonspecific alkaline phosphatase, H, n=7 h-MSCs and n=3 AML-MSCs) and OPN (osteopontin, I, n=6 h-MSCs and n=3 AML-MSCs), and pro-inflammatory gene PTGS2 (prostaglandin-endoperoxide synthase 2, J, n=7 h-MSCs and n=5 AML-MSCs) in h-MSCs pre-treated or not for 72 hours with Ouabain or K + gluconate and in AML-MSCs. T-test was used to compare treated or AML-MSCs groups versus h-MSCs. All histograms show mean ± SEM; * p -value <0.05, ** p -value <0.01, *** p -value <0.001.

Article Snippet: IL-3-dependent 32D murine hematopoietic precursors cells (DSMZ) were cultured for 72 hours on a layer of depolarized or hyperpolarized MSCs, as previously described .

Techniques: Cell Culture, Control, Derivative Assay, Expressing, Co-Culture Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Leukemic Cells Manipulate MSCs Bioelectrical Signals to Reshape the Bone Marrow Niche

doi: 10.1101/2025.03.10.642319

Figure Lengend Snippet: (A) Cell proliferation measured by Presto Blue assay in h-MSCs and AML-MSCs treated or not with 10 µM Lubi or 1 µM IVM for 72 hours (n=4, t-test comparing all groups versus AML-MSCs). (B-C) Cell density of murine IL-3–dependent 32D cell line cultured in the presence or absence of IL-3, with h-MSCs, and compared with 32D cell line co-cultured on AML-MSCs layer treated with Lubi (B, n=5) or IVM (C, n=5). T-test was performed to compare treated groups or h-MSCs versus AML-MSCs, with ±IL3 used as experimental control. (D) Total branches length of HUVEC tubes by using conditioned medium derived from h-MSCs (n=4) and AML-MSCs untreated (n=7) or pre-treated with Lubi (n=8) or IVM (n=5) for 72 hours and then stimulated (st) or not (unst) with a pro-inflammatory cytokine cocktail for 24 hours; tube formation was evaluated after 4 hours and normalized to unst condition (AU: arbitrary unit, t-test comparing treated or h-MSCs stimulated groups versus stimulated AML-MSCs). (E) Percentage of PHA-stimulated CD3 + T cells expressing CD69 and CD25 after 72 hours of co-culture on a layer of h-MSCs or AML-MSCs treated or not with Lubi (10 µM) or IVM (1 µM), relative to SF condition (n=4, t-test comparing treated or h-MSCs groups versus AML-MSCs). (F-G) Relative expression of osteoprogenitor-associated genes TNAP (F, n=7 AML-MSCs, n=4 h-MSCs) and OPN (G, n=6 AML-MSCs, n=4 h-MSCs) in h-MSCs and AML-MSCs treated or not for 72 hours with Lubi (10 µM) or IVM (1 µM) and measured by RQ-PCR (t-test comparing treated or h-MSCs groups versus AML-MSCs). All histograms show mean ± SEM; * p -value <0.05, ** p -value <0.01, *** p -value <0.001, **** p -value <0.0001.

Article Snippet: IL-3-dependent 32D murine hematopoietic precursors cells (DSMZ) were cultured for 72 hours on a layer of depolarized or hyperpolarized MSCs, as previously described .

Techniques: Cell Culture, Control, Derivative Assay, Expressing, Co-Culture Assay

Journal: Cancer Research

Article Title: Oncogenic Calreticulin Induces Immune Escape by Stimulating TGF-β Expression and Regulatory T Cell Expansion in the Bone Marrow Microenvironment

doi: 10.1158/0008-5472.can-23-3553

Figure Lengend Snippet: Figure 1. Mutant CALR induces TGFβ production. A, Heat map showing expression profile of MPL-CALRins5-transduced 32D cells vs. MPL-CALRWT. Relative expression of selective immune regulatory cytokines is plotted. Color codes represent the Z-score log2 intensity. RNA was harvested from four independent IL3-starved cell cultures. Differential gene expression analysis was performed with the linear model–based approach (limma R package). B, Scatter plot showing fold change of mean fluorescence intensity (MFI) from L-TGFβ1 expressed on MPL-CALR-/WT/del52-transduced 32D cells after IL3 withdrawal. Four independent experiments were performed and the results were pooled. P values were calculated using one-way ANOVA. C, Scatter plot showing TGFβ promoter activity (luciferase activity relative to mean of WT control) of 32D-MPL-CalrWT or 32D-MPL-Calrdel52 cells. Cells transfected with the pGL3-TGFβ1 promotor vector. Pooled data from six independent experiments. P values were calculated using the Mann–Whitney test. D and E, Spleens of mice isolated 21 days after injection of either empty vector or CALRdel52-MPL–transduced BM. Exemplary picture (scale in cm; D) and scatter plot (E) showing quantification of weight of spleens of mice 21 days after injection of either empty vector– or CALRdel52-MPL–transduced BM. F–I, scRNA-seq of bone marrow from mice 21 days after injection of either empty vector– (n ¼ 2) or CALRdel52-MPL (n ¼ 2)–transduced BM. UMAP depicting clustering into different cell populations (F), UMAP of empty vector and CALRdel52-MPL condition merged (G), heat map depicting fraction of cells in each cluster (H), and bubble plot depicting TGFβ1 expression in different clusters combining fraction cells expressing TGFβ1 (%, relative expression to mean >0) and expression within clusters relative to mean expression level across all clusters (I). Red arrow, erythroblast population in the bone marrow of mice that received CALRdel52-MPL BMC. J, Scatter plot showing TGFβ protein expression (fold change of MFI) of CD45+ lineage marker negative cells isolated from JAK2V617F KI mice or littermate controls as indicated. Each data point is a biological replicate (individual mouse). P values were calculated using an unpaired Student t test (E and J).

Article Snippet: The murine myeloid cell line 32D (RRID:CVCL_0118) and the murine pro B cell line Ba/F3 (RRID:CVCL_0161) were purchased from DSMZ and cultured in RPMI 1640 Medium + 10% FCS + 1% penicillin–streptomycin (P/S) + 5-ng/mL mIL3.

Techniques: Mutagenesis, Expressing, Gene Expression, Fluorescence, Activity Assay, Luciferase, Control, Transfection, Plasmid Preparation, MANN-WHITNEY, Isolation, Injection, Marker

Journal: Scientific Reports

Article Title: Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms

doi: 10.1038/s41598-024-53240-8

Figure Lengend Snippet: Chromatin segregation defects in murine CALRdel52 or JAK2V617F 32D cells. ( a ) Example of live-cell image galleries of mitotic JAK2V617F transduced 32D MPL cells with chromatin bridges, lagging chromosomes, or micronuclei (insets). Scale bars 5 µm. ( b ) Plots showing the percentage of chromatin bridges, lagging chromosomes and micronuclei in untreated control (EV), CALRdel52 or JAK2V617F transduced 32D MPL cells, or cells treated with 200 nM doxorubicin or 3 µM MPS1 inhibitor (MPS1i) NMS-P715 (6 to 11 independent color-coded experiments with 100 cells per experiment and condition, the horizontal black lines and dispersion bars indicate means and SD of the means). The significance p -values were obtained with the exact Fisher test (EV control versus mutant cells).

Article Snippet: Murine 32D cells (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium (PAN Biotec, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin–Streptomycin and 10% WEHI culture supernatant at 37 °C with 5% CO2.

Techniques: Control, Dispersion, Mutagenesis

Journal: Scientific Reports

Article Title: Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms

doi: 10.1038/s41598-024-53240-8

Figure Lengend Snippet: CALRdel52 and JAK2V617F mutations weaken the spindle assembly checkpoint. ( a ) Duration of mitotic arrest in 100 ng/ml nocodazole treated control (EV), CALRdel52 and JAK2V617F 32D MPL cells co-expressing H2B-mCherry. The violin superplots show pooled data from three independent (color-coded) experiments with 20 cells per experiment and cell line. The thick colored dots indicate independent experiment means. Horizontal red lines indicate medians, black lines mean, and the blue lines quartiles of the pools. One-Way ANOVA (Kruskal–Wallis with Dunn’s post-test; * p < 0.05; **** p < 0.001). ( b ) Plots show the fraction of cells that exit mitosis at the given time after mitotic entry in the presence of 100 ng/ml nocodazole. Bold lines indicate mean, and SDs are indicated by dotted lines. ( c ) The outcome of the nocodazole-induced mitotic arrest was determined in control (EV) or CALRdel52- or JAK2V617F transduced 32D MPL cells. Cell fates were categorized into four groups: spindle-less mitotic exit with direct chromatin decondensation (grey), cell death during mitotic arrest (red), normal chromatin segregation (green), and abnormal chromatin segregation (violet). Columns indicate the means of three independent experiments with 10 cells each, error bars SDs, and points the individual data points. No differences were found between the control (EV) to both mutants within each category. Two-Way ANOVA with Dunnet post-test. ( d ) Duration of mitotic arrest induced with 100 ng/ml nocodazole in the presence or absence of 1 µM ruxolitinib for 4 h in control (EV), CALRdel52 and JAK2V617F 32D MPL cells co-expressing H2B-mCherry. The violin blots show data from 20 random cells (grey dots) per cell line and treatment. Horizontal red lines indicate medians and blue lines quartiles. (One-Way ANOVA (Kruskal–Wallis with uncorrected Dunn’s post-test; * p < 0.05; **** p < 0.001). ( e ) Plots show the fraction of cells treated with 100 ng/ml nocodazole that exit mitosis at the given time after mitotic entry in absence (dotted lines) or presence (bold lines) of 1 µM ruxolitinib.

Article Snippet: Murine 32D cells (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium (PAN Biotec, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin–Streptomycin and 10% WEHI culture supernatant at 37 °C with 5% CO2.

Techniques: Control, Expressing

Journal: Scientific Reports

Article Title: Calreticulin and JAK2V617F driver mutations induce distinct mitotic defects in myeloproliferative neoplasms

doi: 10.1038/s41598-024-53240-8

Figure Lengend Snippet: CALRdel52 and JAK2V617F mutations disturb kinetochore recruitment of SAC factors. Quantitative immunofluorescence for indicated SAC factors at kinetochores in 200 ng/ml nocodazole arrested control (EV), CALRdel52 or JAK2V617F transduced 32D MPL cells stably expressing H2B-mCherry. ( a )–( h ) Representative immunofluorescences of SAC factors (green in overlay) and the kinetochore marker CREST (red in overlay). Violin superplots show the signal ratios of the different SAC factors to CREST at kinetochores as distribution of the pooled data from 3 or 4 independent experiments with n cells per condition as indicated. The horizontal black lines show medians, horizontal red lines the means, blue dotted lines quartiles, and dispersion bars the SDs of the independent experiments. The mean of each experiment is shown as a color-coded dot. The significance p-values were obtained with two-tailed t-test over the means of the independent experiments.

Article Snippet: Murine 32D cells (DSMZ, Braunschweig, Germany) were cultured in RPMI-1640 medium (PAN Biotec, Aidenbach, Germany) supplemented with 10% fetal calf serum (FCS) and 1% Penicillin–Streptomycin and 10% WEHI culture supernatant at 37 °C with 5% CO2.

Techniques: Immunofluorescence, Control, Stable Transfection, Expressing, Marker, Dispersion, Two Tailed Test