cell culture 32d  (DSMZ)

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

Journal: Journal of Biological Chemistry

Article Title: Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling

doi: 10.1074/jbc.m110.205021

Figure Lengend Snippet: FIGURE 1. Down-regulation of DEP-1 in 32D cells results in enhanced FLT3 signaling and enhanced phosphorylation of FLT3.A, whole cell lysates of 32D cellsexpressingwildtypemuFLT3andDEP-1-specificshRNA(targetsA2orA3)oranontargetingcontrolshRNAweresubjectedtoSDS-PAGE,blottedtoaPVDF membrane, and analyzed with antibodies recognizing DEP-1 and vinculin. Chemiluminescence signals were detected using a CCD camera-based chemilumi- nescence detection system and calculated relative to vinculin. Relative (rel.) levels of DEP-1 normalized to vinculin are indicated. B, quantitative RT-PCR for DEP-1 mRNA in the above cell lines. Mean S.D. of at least three independent experiments. C–F, indicated cell lines were starved for 4 h in serum- and cytokine-freemediumandwerestimulatedwithFL(50ng/ml)fortheindicatedtimeperiods.C–H,activationofERK(CandE),AKT(C),STAT5(D),orPLC(G)was analyzed using the indicated phospho-specific antibodies. Blots were reprobed for total signaling proteins. -Actin was analyzed as loading control. Numbers above the phospho-specific blots represent quantification of the phosphor-specific signals, normalized to the corresponding signals with pan-specific anti- bodies, and relative to the signal in unstimulated cells harboring control shRNA, which was set to 1.0. The blots shown are representative for at least three experiments with consistent results. F, comparison of phosphorylation of ERK1/2 detected by conventional anti-pThr-202/pTyr-204 antibodies, and anti-pThr- 202 antibodies. Ratio between ERK1/2 phosphorylation of shDEP-1 to shControl cells of three independent experiments is shown. H, FLT3 was immunopre- cipitated and analyzed by immunoblotting with anti-FLT3, phosphotyrosine (pY100), or phospho-FLT3 (pY591) antibodies. HM, high mannose; CG, complex glycosylated FLT3. Quantification refers to complex glycosylated (surface) FLT3. Representative blots of at least three time repeated experiments are shown.

Article Snippet: Cell Lines, Antibodies, and Antisera—The IL-3-dependent murinemyeloid cell line 32D clone 3 (32D) (GermanCollection 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 1 ng/ml IL-3 or conditioned medium obtained from murine IL-3 producing BPV cells (20).

Techniques: Phospho-proteomics, Membrane, Quantitative RT-PCR, Control, shRNA, Comparison, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling

doi: 10.1074/jbc.m110.205021

Figure Lengend Snippet: FIGURE 2. Down-regulation of DEP-1 causes hyperphosphorylation of human FLT3. 32D cells expressing human FLT3- and DEP-1-specific shRNAs A2 and A3 or a control shRNA were starved and stimulated with FL (50 ng/ml) for indicated time periods. FLT3 was immunoprecipitated and analyzed by immuno- blotting with anti-FLT3 or phosphotyrosine (4G-10, A) or phospho-FLT3 (pY591, B) antibodies. HM, high mannose; CG, complex glycosylated FLT3; UB, ubiq- uitinated. Graphs demonstrate specific phosphorylation of total FLT3 receptor. Phosphospecific to total FLT3 receptor chemiluminescence signals are shown (sum of high mannose, complex glycosylated FLT3, and UB protein). Means S.D. are of at least three independent experiments.

Article Snippet: Cell Lines, Antibodies, and Antisera—The IL-3-dependent murinemyeloid cell line 32D clone 3 (32D) (GermanCollection 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 1 ng/ml IL-3 or conditioned medium obtained from murine IL-3 producing BPV cells (20).

Techniques: Expressing, Control, shRNA, Immunoprecipitation, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling

doi: 10.1074/jbc.m110.205021

Figure Lengend Snippet: FIGURE4.OverexpressionofDEP-1impairsFLT3signalingandphosphor- ylation. 32D cells expressing huFLT3, stably transduced with an expression construct for human DEP-1 or the corresponding control vector, were ana- lyzed for DEP-1 expression, FL-mediated signaling activity, and receptor phosphorylation. A, whole cell lysates were blotted with antibodies recogniz- ing both endogenous murine and exogenous human DEP-1 and vinculin for control (Con). B, expression of transduced huDEP-1 was detected by labeling with a monoclonal antibody recognizing a surface epitope of human DEP-1 and subsequent labeling with anti-mouse-Cy3 antibody. Surface-localized DEP-1 was quantified by flow cytometry. C and D, indicated cell lines were starved for 4 h in serum- and cytokine-free medium and were stimulated with FL (50 ng/ml) for the indicated time periods. Whole cell lysates were blotted andactivationofERKorAKTwasanalyzedusingphospho-specificantibodies. Blots were reprobed for total signaling proteins. D, phosphorylation of FLT3 was analyzed by immunoblotting with anti-phosphotyrosine antibody 4G-10 and anti-phospho FLT3 antibody pTyr-591, and blots were subsequently rep- robed for total FLT3. HM, high mannose; CG, complex glycosylated FLT3. Representative blots of experiments repeated at least three time are demonstrated.

Article Snippet: Cell Lines, Antibodies, and Antisera—The IL-3-dependent murinemyeloid cell line 32D clone 3 (32D) (GermanCollection 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 1 ng/ml IL-3 or conditioned medium obtained from murine IL-3 producing BPV cells (20).

Techniques: Expressing, Stable Transfection, Transduction, Construct, Control, Plasmid Preparation, Activity Assay, Phospho-proteomics, Labeling, Flow Cytometry, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Protein-tyrosine Phosphatase DEP-1 Controls Receptor Tyrosine Kinase FLT3 Signaling

doi: 10.1074/jbc.m110.205021

Figure Lengend Snippet: FIGURE 6. Depletion of DEP-1 stimulates proliferation and clonal growth of 32D cell lines. A, 32D cells expressing wild type muFLT3 and DEP-1 shRNA A2 or control shRNA were seeded in 96-well plates and cell growth in the absence of cytokines, in the presence of FL (20 ng/ml), or in the presence of IL-3 (2 ng/ml) and was measured after 2 days using the MTT method. B, cells were seeded in 96-well plates at a density of 0.8 105 per ml in RPMI 1640 medium supplemented with 10% fetal calf serum without cytokines, with FL (20 ng/ml), or IL-3 (2 ng/ml) as indicated. Cell growth was measured at the indicated time points by measuring cellular GFP fluorescence. Values are means of triplicates, and the shown data set is representative for three experiments with consistent results. C, clonal growth of 32D cells in methylcellulose. The above cell pools were subjected to colony formation assays in methylcellulose, in cytokine in the absence or presence of FL (20 ng/ml) or IL-3 (2.5 ng/ml). Representative sections were photographed after 12 days (FL samples) or 6 days (IL-3 samples) of culture, and colonies were quantified by counting representative fields of the wells.

Article Snippet: Cell Lines, Antibodies, and Antisera—The IL-3-dependent murinemyeloid cell line 32D clone 3 (32D) (GermanCollection 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 1 ng/ml IL-3 or conditioned medium obtained from murine IL-3 producing BPV cells (20).

Techniques: Expressing, shRNA, Control, Fluorescence