Journal: Leukemia

Article Title: A point mutation of zebrafish c-cbl gene in the ring finger domain produces a phenotype mimicking human myeloproliferative disease

doi: 10.1038/leu.2015.154

Figure Lengend Snippet: Increase in HSPCs was dependent on the Flt3 pathway. (a–d) flt3 MO injection attenuated the phenotype of LDD731. (e and f) c-myb staining of zebrafish embryos after treatment with 500 nM Lestaurtinib. (g and h) c-myb staining of embryos after treatment with 100 nM PKC412. (i) CBL mutation decreased FLT3 ubiquitination levels and upregulated mature FLT3. 293T cells were transfected with FLT3 and/or c-CBL constructs as indicated. After lysis, FLT3 was immunoprecipitated, blotted and probed with anti-FLT3 and anti-FK2. (j) Inhibitors were used to treat 293T cells. The cells transfected with FLT3 and c-CBL were treated with or without PKC412 or Lestaurtinib for 24 h. After lysis, protein samples were blotted and probed with FLT3, c-CBL and pFLT3 antibodies.

Article Snippet: FLT3 (fms-like tyrosine kinase 3) inhibitor PKC412 (ref. 22 ) (sc-200691) and Lestaurtinib 23 (sc-218657) with stock concentrations of 10 mM, were purchased from Santa Cruz Biotechnology and stored at − 80 °C.

Techniques: Injection, Staining, Mutagenesis, Ubiquitin Proteomics, Transfection, Construct, Lysis, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Comparison of effects of FLT3 inhibitors against the growth of human AML cell lines. Cells were treated for approximately three days. Targeted SYK inhibitor, PRT062607, was tested in parallel with the FLT3 inhibitors. (A‐F) Treatment of AML cell lines with midostaurin (A), gilteritinib (B), sorafenib (C), crenolanib (D), quizartinib (E) and PRT062607 (F)

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Comparison

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Effects of midostaurin on signalling molecules in wt FLT3‐expressing and mutant FLT3‐expressing cells. (A) Effects of midostaurin at 100 nmol/L on levels of Bcl‐xL, Bcl‐2 and Mcl‐1 in mutant FLT3‐expressing MOLM14 cells. (B, C) Effects of midostaurin on levels of Bcl‐xL, Bcl‐2 and Mcl‐1 in wt FLT3‐expressing Kasumi‐1‐luc+ cells (B) or OCI‐AML2 cells (C). (D) Induction of apoptosis by midostaurin in wt FLT3‐expressing AML cells. The apoptosis studies carried out for midostaurin‐treated Kasumi‐1‐luc+ cells and OCI‐AML2 cells were performed once each. The flow cytometry data from which the numerical values shown in the bar graphs were derived are now shown in Figure . (E) Effects of midostaurin at the indicated concentrations on phosphorylation of key molecules involved in PI3K/AKT and MAPK signalling in wt FLT3‐expressing Kasumi‐1‐luc+ cells. (F) Effects of midostaurin at the indicated concentrations on phosphorylation of S6 in wt FLT3‐expressing AML cell lines conferring differential sensitivity to midostaurin. WB = Western blot

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Expressing, Mutagenesis, Flow Cytometry, Derivative Assay, Phospho-proteomics, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: In vivo effects of midostaurin on growth of leukaemia cells in an SKNO‐1‐luc+ xenograft model and in an OCI‐AML3‐luc+ xenograft model. (A) Measure of leukaemia burden in vehicle‐ versus midostaurin‐treated mice for SKNO‐1‐luc+ xenograft model. For total flux bioluminescence, the Mann‐Whitney test (two‐tailed) was carried out. Vehicle versus midostaurin, P < .0001 (Day 8); P < .0001 (Day 15); P < .0001 (Day 22); P < .0001 (Day 29). (B) Measure of survival of vehicle‐ versus midostaurin‐treated mice for SKNO‐1‐luc+ xenograft model. The log‐rank (Mantel‐Cox) test and the Gehan‐Breslow‐Wilcoxon tests were carried out for survival curve comparisons. For both tests, P < .0001. (C) Effects of midostaurin in vivo against mice harbouring SKNO‐1‐luc+ cells: supine and prone (high scale). Days 22‐36. Representative images (n = 5). (D) Measure of leukaemia burden in vehicle‐ versus midostaurin‐treated mice for OCI‐AML3‐luc+ xenograft model. For total flux bioluminescence, the Mann‐Whitney test (two‐tailed) was carried out. Vehicle versus midostaurin (80 mg/kg, Day 15), P = 0656; vehicle versus midostaurin (100 mg/kg, Day 15), P < .0001; midostaurin (80 mg/kg, Day 15) versus midostaurin (100 mg/kg, Day 15), P = .0001. (E) Measure of survival of vehicle‐ versus midostaurin‐treated mice for OCI‐AML3‐luc+ xenograft model. The log‐rank (Mantel‐Cox) test and the Gehan‐Breslow‐Wilcoxon tests were carried out for survival curve comparisons. For both tests, P < .0001. For vehicle versus midostaurin (80 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0547, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0210. For midostaurin (80 mg/kg) versus midostaurin (100 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0027, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0025. For vehicle versus midostaurin (100 mg/kg), the log‐rank (Mantel‐Cox) test yielded a P value = .0001, and the Gehan‐Breslow‐Wilcoxon test yielded a P value = .0002. (F) Effects of midostaurin in vivo against mice harbouring OCI‐AML3‐luc+ cells. Supine and prone (high scale), Days 8, 15 and 22. Representative images (n = 4)

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: In Vivo, MANN-WHITNEY, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Effects of the multi‐kinase inhibitor midostaurin in combination with chemotherapy in models of acute myeloid leukaemia

doi: 10.1111/jcmm.14927

Figure Lengend Snippet: Comparison of effects of FLT3 inhibitors on growth of cells expressing activated SYK or oncogenic FLT3. (A‐C) Proliferation curves generated for parental Ba/F3 cells, Ba/F3‐SYK‐TEL cells and Ba/F3‐FLT3‐ITD cells treated for approximately three days with FLT3 inhibitors (crenolanib, quizartinib, midostaurin, sorafenib and gilteritinib) or the targeted SYK inhibitor, PRT062607. (D) Comparison of effects of FLT3 inhibitors on activation of signalling molecules downstream of activated SYK. PRT062607 was included as a positive control for SYK inhibition

Article Snippet: 80 mg/kg was the tolerated dose for midostaurin purchased from MedChemExpress, LC Labs and BOC Sciences, whereas 100 mg/kg was the tolerated dose for midostaurin received from Novartis Pharma AG.

Techniques: Comparison, Expressing, Generated, Activation Assay, Positive Control, Inhibition