32d german collection of microorganisms and cell cultures dsmz  (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: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) Elevated level of cleaved PARP-1 in 32D p210 cells treated with apoptin. (b) Appearance of cleaved PARP-1 and induction of apoptosis in Bcr-Abl expressing 32D p210 cells when treated with apoptin or imatinib; (c) The effects of apoptin on the survival of Bcr-Abl expressing cells as determined by Nicoletti method. N=3. *P<0.03.

Article Snippet: As shown in figure apoptin derived decapeptide treatment does not show any significant cellular toxicity among 32D DSMZ as compared to control and scrambled peptide treated cells.

Techniques: Expressing

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) . Schematic representation of apoptin deletion mutants tagged with N-terminal GFP. (b) . Immunoblot showing the expression of deletion mutants and wild type apoptin (1–121) transfected into PC-3 cells and immunoprecipitated with anti-GFP antibody 18 h post-transfection. (c) . Apoptin CO-IP experiment from transfected 32D DMSZ , 32D p210 and K562 cells with various mutant forms of apoptin; Bcr-Abl was identified in the immuno-precipitates of full-length apoptin and apoptin derivatives that harbored amino acids from 74-100 (includes the proline rich region, aa: 81-86) indicating a part of this region of apoptin is important for the interaction with Bcr-Abl p210 .

Article Snippet: As shown in figure apoptin derived decapeptide treatment does not show any significant cellular toxicity among 32D DSMZ as compared to control and scrambled peptide treated cells.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32D DSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32D p210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f) . Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.

Article Snippet: As shown in figure apoptin derived decapeptide treatment does not show any significant cellular toxicity among 32D DSMZ as compared to control and scrambled peptide treated cells.

Techniques: Derivative Assay, Expressing, MTT Assay

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) Elevated level of cleaved PARP-1 in 32D p210 cells treated with apoptin. (b) Appearance of cleaved PARP-1 and induction of apoptosis in Bcr-Abl expressing 32D p210 cells when treated with apoptin or imatinib; (c) The effects of apoptin on the survival of Bcr-Abl expressing cells as determined by Nicoletti method. N=3. *P<0.03.

Article Snippet: The above observation was further confirmed by GST-apoptin and BCR-ABL1 p210 ‘pull-down assay’ using both BCR-ABL1-positive (32D p210 ), and -negative (32D DSMZ ) cell lines (Fig. , ) where full length BCR-ABL1 p210 with intact SH3 domain showed interaction with GST-apoptin and were ‘pulled-down’ by glutathione sepharose beads.

Techniques: Expressing

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) . Schematic representation of apoptin deletion mutants tagged with N-terminal GFP. (b) . Immunoblot showing the expression of deletion mutants and wild type apoptin (1–121) transfected into PC-3 cells and immunoprecipitated with anti-GFP antibody 18 h post-transfection. (c) . Apoptin CO-IP experiment from transfected 32D DMSZ , 32D p210 and K562 cells with various mutant forms of apoptin; Bcr-Abl was identified in the immuno-precipitates of full-length apoptin and apoptin derivatives that harbored amino acids from 74-100 (includes the proline rich region, aa: 81-86) indicating a part of this region of apoptin is important for the interaction with Bcr-Abl p210 .

Article Snippet: The above observation was further confirmed by GST-apoptin and BCR-ABL1 p210 ‘pull-down assay’ using both BCR-ABL1-positive (32D p210 ), and -negative (32D DSMZ ) cell lines (Fig. , ) where full length BCR-ABL1 p210 with intact SH3 domain showed interaction with GST-apoptin and were ‘pulled-down’ by glutathione sepharose beads.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32D DSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32D p210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f) . Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.

Article Snippet: The above observation was further confirmed by GST-apoptin and BCR-ABL1 p210 ‘pull-down assay’ using both BCR-ABL1-positive (32D p210 ), and -negative (32D DSMZ ) cell lines (Fig. , ) where full length BCR-ABL1 p210 with intact SH3 domain showed interaction with GST-apoptin and were ‘pulled-down’ by glutathione sepharose beads.

Techniques: Derivative Assay, Expressing, MTT Assay

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) Elevated level of cleaved PARP-1 in 32D p210 cells treated with apoptin. (b) Appearance of cleaved PARP-1 and induction of apoptosis in Bcr-Abl expressing 32D p210 cells when treated with apoptin or imatinib; (c) The effects of apoptin on the survival of Bcr-Abl expressing cells as determined by Nicoletti method. N=3. *P<0.03.

Article Snippet: The murine IL3-dependent primary hematopoietic murine cell line 32D DSMZ was used as the control cell line.

Techniques: Expressing

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) . Schematic representation of apoptin deletion mutants tagged with N-terminal GFP. (b) . Immunoblot showing the expression of deletion mutants and wild type apoptin (1–121) transfected into PC-3 cells and immunoprecipitated with anti-GFP antibody 18 h post-transfection. (c) . Apoptin CO-IP experiment from transfected 32D DMSZ , 32D p210 and K562 cells with various mutant forms of apoptin; Bcr-Abl was identified in the immuno-precipitates of full-length apoptin and apoptin derivatives that harbored amino acids from 74-100 (includes the proline rich region, aa: 81-86) indicating a part of this region of apoptin is important for the interaction with Bcr-Abl p210 .

Article Snippet: The murine IL3-dependent primary hematopoietic murine cell line 32D DSMZ was used as the control cell line.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32D DSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32D p210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f) . Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.

Article Snippet: The murine IL3-dependent primary hematopoietic murine cell line 32D DSMZ was used as the control cell line.

Techniques: Derivative Assay, Expressing, MTT Assay

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) Elevated level of cleaved PARP-1 in 32D p210 cells treated with apoptin. (b) Appearance of cleaved PARP-1 and induction of apoptosis in Bcr-Abl expressing 32D p210 cells when treated with apoptin or imatinib; (c) The effects of apoptin on the survival of Bcr-Abl expressing cells as determined by Nicoletti method. N=3. *P<0.03.

Article Snippet: 32D DSMZ cells are strictly murine IL3-dependent, so the media was supplemented with 10 % media supernatant from WEHI-3B cells [ ].

Techniques: Expressing

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) . Schematic representation of apoptin deletion mutants tagged with N-terminal GFP. (b) . Immunoblot showing the expression of deletion mutants and wild type apoptin (1–121) transfected into PC-3 cells and immunoprecipitated with anti-GFP antibody 18 h post-transfection. (c) . Apoptin CO-IP experiment from transfected 32D DMSZ , 32D p210 and K562 cells with various mutant forms of apoptin; Bcr-Abl was identified in the immuno-precipitates of full-length apoptin and apoptin derivatives that harbored amino acids from 74-100 (includes the proline rich region, aa: 81-86) indicating a part of this region of apoptin is important for the interaction with Bcr-Abl p210 .

Article Snippet: 32D DSMZ cells are strictly murine IL3-dependent, so the media was supplemented with 10 % media supernatant from WEHI-3B cells [ ].

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: Oncotarget

Article Title: Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

doi:

Figure Lengend Snippet: (a) The effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl non-expressing 32D DSMZ cells (MTT assay). (b) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing 32D p210 cells (MTT assay). (c) the effects of Tat-conjugated apoptin derived peptide on the survival of Bcr-Abl expressing K562 cells (MTT assay). (d, e, f) . Both Imatinib responsive and resistant patient samples are sensitive to apoptin derived decapeptide but not healthy donor samples. MTT assay results show a time dependent cell death by apoptin decapeptide at 16h, 36h and 48h. N=3. *p<0.05.

Article Snippet: 32D DSMZ cells are strictly murine IL3-dependent, so the media was supplemented with 10 % media supernatant from WEHI-3B cells [ ].

Techniques: Derivative Assay, Expressing, MTT Assay