murine myeloid cell line 32d  (DSMZ)

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: 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