il 3 dependent 32d murine hematopoietic precursors cells  (DSMZ)

Journal: BMC Biology

Article Title: TRAM-LAG1-CLN8 domain-containing protein TMEM56 regulates cell migration by changing intracellular ceramide levels

doi: 10.1186/s12915-026-02614-7

Figure Lengend Snippet: TMEM56 is critical for cell migration. a Distribution of migration relevant target genes identified in a genome-wide loss of function shRNA screening assay. b Migration capability of murine 32D cells (32D) and 32D cells expressing TMEM56 specific shRNA (clones 1 and 2). All cells were co-transduced with either TMEM56 CDS, GFP control CDS, or expressed no additional CDS (w/o). Mean values and standard deviation (SD) of three independent experiments are given. c Migration potential of TMEM56-depleted human Jurkat T cells. Jurkat cells were transduced with lentiviral vectors expressing either a control shRNA or TMEM56-specific shRNA. Data are presented as percentage values ± SD. Differences among groups were assessed by one-way ANOVA, followed by pairwise t-tests with Bonferroni correction to identify significant differences. Significance is indicated as: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; ns = not significant. d Representative images of zebrafish embryos injected at the 1-cell stage with a pool of the three TMEM56-specific morpholinos (TMEM56 MOs) or co-injected with MO-resistant TMEM56 variant tlcd4b mRNA. The red arrowhead indicates the normal position (main cluster) of the primordial germ cells (PGC) in the embryo; black arrowheads show ectopic positions

Article Snippet: Murine 32D cells (ATCC CRL-11346, non-authenticated, mycoplasma negative) were cultured in RPMI 1640 medium (PAA Laboratories, Pasching, Austria) supplemented with murine recombinant interleukin (IL)−3 (100 ng mL −1 ; R&D Systems, Minneapolis, USA).

Techniques: Migration, Genome Wide, shRNA, Screening Assay, Expressing, Clone Assay, Transduction, Control, Standard Deviation, Injection, Variant 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 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: Cellular Oncology (Dordrecht, Netherlands)

Article Title: BCAT1 contributes to the development of TKI-resistant CML

doi: 10.1007/s13402-024-01003-y

Figure Lengend Snippet: BCAT1 is required for the leukemogenesis of TKI-resistant CML. A The mRNA expression levels of BCAA metabolism-related genes were detected via qRT‒PCR in the BCR-ABL–induced CML (BCR-ABL-CML) model and BCR-ABL T315I -induced TKI-resistant CML (BCR-ABL T315I -CML) model ( n = 3). B Mouse Bcat1 shRNA was constructed, and the knockdown efficiency was verified by qRT‒PCR ( n = 3). C The number of 32Dcl3-BCR-ABL T315I cells was counted on the indicated days after BCAT1 was knocked down by shRNAs (sh Bcat1 #1 and #2) or a scrambled control ( n = 3). D Experimental design. BCR-ABL T315I retroviruses and Bcat1 shRNA lentivirus were infected into 32Dcl3-BCR-ABL T315I cells, which were subsequently sorted by flow cytometry and transplanted into sublethally irradiated C3H/HeN recipient mice. E Flow cytometric analysis of leukemia cells in the peripheral blood of recipients transplanted with Bcat1 -knockdown (sh Bcat1 #1 and #2) 32Dcl3-BCR-ABL T315I cells or scrambled shRNA ( n = 5). F Quantification of the weights of the spleens and livers of recipients transplanted with Bcat1 -knockdown (sh Bcat1 #1 and #2) 32Dcl3-BCR-ABL T315I cells or scrambled shRNA ( n = 3). G Survival data for recipient mice (sublethally irradiated) transplanted with Bcat1 -knockdown (sh Bcat1 #1 and #2) 32Dcl3-BCR-ABL T315I cells or scrambled shRNA ( n = 5). The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The murine IL-3-dependent myeloid cell Line 32Dcl3 and the Imatinib-resistant cell Line K562 R were obtained from ATCC (CRL-11346 and CRL-3344, respectively).

Techniques: Expressing, shRNA, Construct, Knockdown, Control, Infection, Flow Cytometry, Irradiation

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: BCAT1 contributes to the development of TKI-resistant CML

doi: 10.1007/s13402-024-01003-y

Figure Lengend Snippet: BCAT1 enhances leukemogenesis in TKI-resistant CML by activating CREB phosphorylation. A The phospho-CREB and total CREB levels were evaluated in 32Dcl3-BCR-ABL T315I cells after infection with shRNAs targeting Bcat1 (sh Bcat1 #1 and #2) or scrambled shRNA by immunoblotting ( n = 3). B Phospho-CREB and total CREB levels were evaluated in 32Dcl3-BCR-ABL T315I cells upon BCAA treatment by immunoblotting in a chase experiment ( n = 3). C Phospho-CREB and total CREB levels were measured by immunoblotting in 32Dcl3-BCR-ABL T315I cells after treatment with the indicated doses of gabapentin ( n = 3). D The numbers of scramble, sh Bcat1 #1 or sh Bcat1 #2 32Dcl3-BCR-ABL T315I cells overexpressing empty vector (EV) or CREB were counted on the indicated days ( n = 3). E, F The leukemia cell (mCherry + ) frequencies in the peripheral blood ( E ) ( n = 5) and overall survival ( F ) ( n = 5) were compared among the recipients transplanted with scramble, sh Bcat1 #1, sh Bcat1 #2, CREB-overexpressing scramble, sh Bcat1 #1 or sh Bcat1 #2 32Dcl3-BCR-ABL T315I cells. G The expression levels of phospho-CREB and total CREB in scramble, sh Bcat1 #1, sh Bcat1 #2, CREB-overexpressing scramble, sh Bcat1 #1 and sh Bcat1 #2 leukemia cells were determined via western blotting ( n = 3). The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The murine IL-3-dependent myeloid cell Line 32Dcl3 and the Imatinib-resistant cell Line K562 R were obtained from ATCC (CRL-11346 and CRL-3344, respectively).

Techniques: Phospho-proteomics, Infection, shRNA, Western Blot, Plasmid Preparation, Expressing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: BCAT1 contributes to the development of TKI-resistant CML

doi: 10.1007/s13402-024-01003-y

Figure Lengend Snippet: A BCAA dietary restriction inhibits the development of TKI-resistant CML. A The experimental procedure for testing the effect of a BCAA dietary restriction on the development of imatinib-resistant CML. BCR-ABL T315I -CML cells or 32Dcl3-BCR-ABL T315I cells were transplanted into C57BL/6 or C3H/HeN recipient mice, which were subsequently administered a normal BCAA diet (NBCAA) or a low-BCAA (LBCAA) diet for analyses. B, C 32Dcl3-BCR-ABL T315I was transplanted into C3H/HeN mice, and the percentage of GFP + leukemia cells in peripheral blood was analyzed ( B ) by flow cytometry ( A ) after feeding with low BCAA or normal BCAA ( n = 5). D Representative images of the sizes of the spleens and livers of recipients fed low BCAA or normal BCAA diets. E Quantification of the data in Panel C ( n = 3). F Survival curve of recipients transplanted with 32Dcl3-BCR-ABL T315I and fed low or normal BCAA ( n = 5). G Primary BCR-ABL T315I CML cells were transplanted into recipients and fed a low concentration of BCAA or normal BCAA. The weights of the liver and spleen of the recipient mice were evaluated ( n = 3). H Survival analysis of recipients transplanted with BCR-ABL T315I CML cells and fed low or normal BCAA ( n = 5). The data are presented as the means ± SDs. * P < 0.05, ** P < 0.01. I A model showing how BCAT1 regulates the development of TKI-resistant CML. BCAT1 controls the anabolism of BCAA, which further enhances leukemogenesis by activating CREB phosphorylation

Article Snippet: The murine IL-3-dependent myeloid cell Line 32Dcl3 and the Imatinib-resistant cell Line K562 R were obtained from ATCC (CRL-11346 and CRL-3344, respectively).

Techniques: Flow Cytometry, Concentration Assay, Phospho-proteomics

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

Journal: Frontiers in Oncology

Article Title: Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD

doi: 10.3389/fonc.2022.1017947

Figure Lengend Snippet: Phosphorylation of FLT3 in MV4-11, THP-1, and 32D FLT3 ITD PTPRJ KO AML cell lines stably re-expressing PTPRJ wt or TMD G979L, G983L, or G987L mutants. Cells were starved for 4 h in serum-free medium and lysed in RIPA buffer. During starvation, MV4-11 and 32D FLT3 ITD cells were treated with DPI (0.5 µM) or AC220 (20 nM), as indicated. THP-1 cells were stimulated with FL (200 ng/ml, 5 min) before harvest. FLT3 was enriched from 100 – 180 µg lysate by wheat germ agglutinin precipitation and subjected to SDS-PAGE and immunoblotting. Blots were first probed by phospho-site specific antibodies recognizing FLT3 pY591 and re-probed for total FLT3. (A–C) Left: Representative immunoblots (n = 3) are shown. Positions of immature, high mannose (HM, 130 kDa) and mature, complex glycosylated (CG, 160 kDa) forms of FLT3 are indicated by arrows. Positions of 130 and 180 kDa molecular weight standard bands are shown on the left side of the blots. 79 – G979L; 83 – G983L; 87 – G987L. Right : Quantification of specific FLT3 phosphorylation. Values were calculated as the ratio of phosphorylated to total receptor (sum of HM and CG signal) and normalized to DPI-treated or FL-stimulated wt. All values are given as mean ± standard deviation, n = 3. Statistics: one-way ANOVA, followed by Dunnett’s multiple comparisons tests; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 compared to wt.

Article Snippet: The IL-3-dependent murine myeloid cell line 32D clone 3 (32D) (German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany) was maintained in RPMI 1640 medium supplemented with sodium pyruvate (5 mg/ml), 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), and IL-3 (1 ng/ml).

Techniques: Phospho-proteomics, Stable Transfection, Expressing, SDS Page, Western Blot, Molecular Weight, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD

doi: 10.3389/fonc.2022.1017947

Figure Lengend Snippet: Signaling analysis of THP-1 (A) MV4-11 (B) and 32D FLT3 ITD cells expressing TMD mutant PTPRJ. (A) THP-1 PTPRJ KO cells or cells re-expressing PTPRJ wt, G979L, or G983L were starved for 4 h in serum-free medium, then stimulated with 200 ng/ml FLT3 ligand for 5 min (+FL) or left unstimulated (–FL) and lysed in RIPA buffer. (B, C) MV4-11 and 32D FLT3 ITD PTPRJ KO cells or cells re-expressing PTPRJ wt, G979L, G983L, or G987L were starved for 4 h in serum-free medium and treated with DPI (0.5 µM), AC220 (20 nM), or mock (DMSO), as indicated, then lysed in RIPA buffer. Equivalent amounts of protein samples were separated by SDS-PAGE and blotted to a nitrocellulose membrane. Blots were first probed by phospho-site specific antibodies recognizing pSTAT5 (Y694), pAKT (S473), and pERK1/2 (T202/Y204). Blots were re-probed for total STAT5, AKT, and ERK1/2 and subsequently analyzed with antibodies recognizing vinculin (124 kDa) and beta-actin (42 kDa) as a loading control. Left : Representative immunoblots are shown. Dashes indicate positions of molecular weight standard bands. 79 – G979L; 83 – G983L; 87 – G987L Right : Quantification of specific phosphorylation of AKT, ERK1/2, and STAT5 in relation to the total protein level. Values were normalized to FL-stimulated or DPI treated wt and are given as mean ± standard deviation, n = 3 - 4. Statistics: two-way ANOVA and Dunnett’s multiple comparisons test; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 compared to respective wt.

Article Snippet: The IL-3-dependent murine myeloid cell line 32D clone 3 (32D) (German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany) was maintained in RPMI 1640 medium supplemented with sodium pyruvate (5 mg/ml), 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), and IL-3 (1 ng/ml).

Techniques: Expressing, Mutagenesis, SDS Page, Membrane, Control, Western Blot, Molecular Weight, Phospho-proteomics, Standard Deviation

Journal: Frontiers in Oncology

Article Title: Disrupting PTPRJ transmembrane-mediated oligomerization counteracts oncogenic receptor tyrosine kinase FLT3 ITD

doi: 10.3389/fonc.2022.1017947

Figure Lengend Snippet: Clonal growth of FLT ITD cells expressing TMD mutant PTPRJ proteins. MV4-11 and 32D FLT3 ITD PTPRJ KO cells re-expressing PTPRJ wt, G979L, G983L, or G987L were seeded in triplicates in 1.27% methylcellulose-containing, cytokine-free medium. Colonies were stained with iodonitrotetrazolium chloride after 7 days (32D FLT3 ITD) or 10 days incubation (MV4-11) and quantified using Image (J) (A) Number (#) of colonies per well relative (rel) to wt is shown. Values are given as mean ± standard deviation, n = 3. Statistics for MV4-11: one-way ANOVA and Dunnett’s multiple comparisons test; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001 compared to respective wt (ns; not significant). Statistics for 32D FLT3 ITD: unpaired two-tailed t-test; * p ≤ 0.05. (B) Representative images of whole wells are shown. 79 – G979L; 83 – G983L; 87 – G987L.

Article Snippet: The IL-3-dependent murine myeloid cell line 32D clone 3 (32D) (German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany) was maintained in RPMI 1640 medium supplemented with sodium pyruvate (5 mg/ml), 10% heat-inactivated fetal calf serum (FCS), L-glutamine (2 mM), and IL-3 (1 ng/ml).

Techniques: Expressing, Mutagenesis, Staining, Incubation, Standard Deviation, Two Tailed Test