baf3 flt3 itd cells  (TargetMol)

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: Ningetinib significantly inhibits the activities of FLT3-ITD AML cells. A. Dose‒response curves for the FLT3-WT and FLT3-ITD (MV4-11 and MOLM13) AML cell lines after treatment with ningetinib for 48 h. The mean viability of triplicate at concentration 0 was normalized as 100% as control. The data are representative of three experiments. B. IC 50 values for ningetinib for the FLT3-WT (K562, HL60, OCI-AML2, OCI-AML3, U937, and THP-1) and FLT3-ITD (MV4-11 and MOLM13) AML cell lines. C, D. Proportion of apoptotic cells among the MV4-11 and MOLM13 cells treated with different concentrations of ningetinib for 48 h. E, F. Expression of PARP1, caspase 8, cleaved PARP1 and cleaved caspase 8 in MV4-11 and MOLM13 cells as analyzed by western blot after 48 h of treatment with different concentrations of ningetinib. G, H. Percentage of cells in different cell cycle phases detected by flow cytometry after treating cells with DMSO or 3 nM ningetinib for 24 h. I. Dose‒response curves for Ba/F3 and Ba/F3-FLT3-ITD cells treated with ningetinib for 48 h. The mean viability of triplicate at concentration 0 was normalized as 100% as control. The data are representative of three experiments

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: Concentration Assay, Control, Expressing, Western Blot, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: Ningetinib inhibits FLT3 phosphorylation and exhibits antitumor activity against FLT3-ITD mutation in vivo. A, B, C. Western blot analysis of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK and ERK in the MV4-11 and MOLM13 cells after treatment with ningetinib at the indicated doses for 2 h or 6 h. D. Schematic representation of the leukemic mouse model induced by Ba/F3-FLT3-ITD cells. E. Schematic representation of xenograft experiments using human MOLM13 cells. F. Proportion of GFP-positive cells in the BM and SP of mice, measured by flow cytometry on Day 12 ( n = 3 mice per group). G. The survival curves of BaF3-FLT3-ITD-diseased mice treated with the vehicle ( n = 7), ningetinib ( n = 7), gilteritinib ( n = 7) or quizartinib ( n = 7). H. The percentage of human CD45 positive cells in BM and SP of MOLM13-diseased NSG mice detected by flow cytometry on day 22 ( n = 3 mice per group). Error bars indicate mean ± standard error, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: Phospho-proteomics, Activity Assay, Mutagenesis, In Vivo, Western Blot, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: The binding of ningetinib to FLT3. A. Overview of the docking results for ningetinib and FLT3 (Protein Data Bank: 6JQR) from two orthogonal views. B. Detailed docking sites for the binding of ningetinib and FLT3. Proteins are shown as cartoons, active pockets are shown as surfaces, green sticks are interacting amino acid residues, white sticks are small molecules, and orange dashed lines are hydrogen bonds. C. Ba/F3-FLT3-ITD cells were treated with ningetinib (10 µM) or DMSO for 1 h. The temperature ranged from 37 to 55 °C for testing. D. Proteins were quantified using ImageJ, and melting curves for FLT3 were plotted. The data are presented as means ± standard errors from three independent experiments

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: Ningetinib inhibits secondary resistant TKD mutations in vitro. A. Dose‒response curve for cells with secondary mutations (FLT3-ITD-D835Y/D835V/Y842C/N676D/F691L) after treatment with ningetinib for 48 h. The mean viability of triplicate at concentration 0 was normalized as 100% as control. Error bars indicate the mean ± standard error, n = 3 independent experiments. B, C, D, E. Western blot analysis of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK and ERK expression in the FLT3-ITD-D835Y/D835V/Y842C/F691L cells treated with ningetinib for 6 h. Tubulin was used as a loading control

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: In Vitro, Concentration Assay, Control, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: Ningetinib exerts efficient inhibitory effects on FLT3-ITD-F691L mutations in vivo. A. Schematic representation of the mouse model of leukemia induced by Ba/F3-FLT3-ITD-F691L cells. B. Percentage of GFP-positive cells in PB of BALB/c mice, measured by flow cytometry on Day 10 ( n = 6 mice per group). C, D. Proportions of GFP-positive cells in BM and SP of BALB/c mice, examined by euthanizing 3 mice per group on Day 12. E. Weights of the spleens of the sacrificed mice in C ( n = 3 per group). F. Images of the spleens of sacrificed mice in E. G Images of the H&E-stained mouse livers and spleens. H. Survival curves for BaF3-FLT3-ITD-F691L diseased mice treated with the vehicle ( n = 6), ningetinib ( n = 6), gilteritinib ( n = 6) or quizartinib ( n = 6). I. Schematic representation of xenograft experiments using human MOLM13-FLT3-ITD-F691L cells. J. The percentage of human CD45 positive cells in BM and SP of MOLM13-FLT3-ITD-F691L diseased NSG mice detected by flow cytometry on day 22 ( n = 3 mice per group). Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: In Vivo, Flow Cytometry, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Ningetinib, a novel FLT3 inhibitor, overcomes secondary drug resistance in acute myeloid leukemia

doi: 10.1186/s12964-024-01729-0

Figure Lengend Snippet: Ningetinib exhibits therapeutic potential in primary cells derived from patients with FLT3-ITD mutations. A, B, C, D. Cell viability values for primary BM cells from AML patients with FLT-ITD or FLT3-WT after treatment with different concentrations of ningetinib. The data are representative of three experiments. E. Cell viability values for PBMCs from healthy donors after treatment with different concentrations of ningetinib. F, G. Primary cells from FLT3-ITD-positive AML patients were cultured with ningetinib for 12 h. Proteins were extracted, and the expression of p-FLT3, FLT3, p-STAT5, STAT5, p-AKT, AKT, p-ERK and ERK was assessed by western blot analysis. Data are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: Briefly, 1 × 10 7 BaF3- FLT3-ITD cells in 10-cm dishes were incubated with ningetinib (10 μM) or DMSO for 60 min, and then, the cells were collected, washed, resuspended in PBS supplemented with protease inhibitors (TargetMol) and evenly divided into 6 portions for heat treatment at 37–54 °C for 3 min. Proteins were extracted using liquid nitrogen and quantified by immunoblotting.

Techniques: Derivative Assay, Cell Culture, Expressing, Western Blot