Journal: Leukemia

Article Title: Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors

doi: 10.1038/leu.2014.179

Figure Lengend Snippet: Cell surface markers of REtr and REtr+c-KIT(N822K) cells after outgrowth. ( a ) Multi-parameter FACS blots illustrating the differentiation status of REtr- and REtr+c-KIT(N822K)-expressing cells after outgrowth. ( b ) Wright–Giemsa staining and CD34/CD38 FACS blots of REtr and REtr+c-KIT(N822K) cells. ( c ) Cell morphology distribution of REtr and REtr+c-KIT(N822K) cells (representative values). ( d ) Generation of progenitor cells carrying floxed REtr (REtrflox) and c-KIT(N822K) after outgrowth. ( e ) Growth kinetics of controls and Cre/YFP-expressing REtr flox +c-KIT(N822K) cells. ( f ) Differentiation profile and DRAQ5 cell cycle distribution following REtr flox excision at day 8 after Cre-T2A-YFP transduction.

Article Snippet: For cell cycle analysis, cells were incubated for 15 min with 2 μ M DRAQ5 (Alexis Biochemicals, San Diego, CA, USA) at 37 °C followed by FACS analysis.

Techniques: Expressing, Staining, Transduction

Journal: Leukemia

Article Title: Activating c-KIT mutations confer oncogenic cooperativity and rescue RUNX1/ETO-induced DNA damage and apoptosis in human primary CD34+ hematopoietic progenitors

doi: 10.1038/leu.2014.179

Figure Lengend Snippet: Cell cycle analysis and apoptosis in REtr and REtr+c-KIT(N822K) cells after outgrowth. ( a ) Cell cycle analysis of mixed cell populations at day 10 after transduction of CD34+ cells. Cells were stained with DRAQ5 and measured by FACS. ( b ) Cell cycle analysis (DRAQ5) after outgrowth of REtr- and REtr+c-KIT(N822K)-expressing cells. ( c ) Level of apoptosis in REtr- and REtr+c-KIT(N822K)-expressing cells by Annexin-V staining, subG1 (DRAQ5) and ZVAD-FITC staining. ( d ) Western blotting analysis of apoptosis-relevant proteins in REtr- and REtr+c-KIT(N822K)-expressing cells after outgrowth.

Article Snippet: For cell cycle analysis, cells were incubated for 15 min with 2 μ M DRAQ5 (Alexis Biochemicals, San Diego, CA, USA) at 37 °C followed by FACS analysis.

Techniques: Cell Cycle Assay, Transduction, Staining, Expressing, Western Blot

Journal:

Article Title: A KIT juxtamembrane PY567 -directed pathway provides nonredundant signals for erythroid progenitor cell development and stress erythropoiesis

doi: 10.1016/j.exphem.2008.10.009

Figure Lengend Snippet: (A) Ex vivo growth profiles are illustrated for bone marrow–derived (pro)erythroblasts from Kit+/+, KitY567F/Y567F, and Lyn−/− mice. Kitpos progenitors (as isolated from bone marrow of KitY567F/Y567F and congenic control mice) were cultured in SP34-ex media in the presence of stem cell factor (SCF; 100 ng/mL) and erythropoietin (Epo; 2.5 U/mL). Viable cells counts were performed daily. Note the marked defect in expansion potential of KitY567F/Y567F erythroblasts. (B) Left panel: bone marrow–derived progenitor cells were prepared from wild-type Kit+/+ (wt-KIT) and KitY567F/Y567F mice, and expanded in SP34-ex medium. At day 5, frequencies of Annexin-V positive vs negative cells among KitposCD71high and KitnegCD71high erythroblasts were analyzed. (B) Right panel: For splenic erythroblasts produced in vivo in response to phenylhydrazine-induced anemia, substantial increases in frequencies of Annexin-V–positive apoptotic erythroblasts also were observed. (C) Left panel: At day 3.5 post-phenylhydrazine treatment of KitY567F/Y567F and Kit+/+ mice, BrdU was injected (tail vein). At 1.5 hours spleens were isolated. Post Ter119pos cell depletion, CD71high erythroid progenitors were retrieved via magnetic-activated cell sorting, and frequencies of S-phase (proliferating) CD71high erythroid progenitors were estimated via flow cytometry. (C) Right panel: At day 3.5 post-phenylhydrazine treatment of KitY567F/Y567F and Kit+/+ mice, erythroid progenitors from spleen were prepared and cultured in the presence of KIT-L at 50 ng/mL (and Epo at 2.5 U/mL). At 16 hours of culture, cells were stained with YoPro1 to determine cell survival. (D) To test whether defects in KitY567F/Y567F erythroblast expansion are exhibited during Epo-induced erythropoiesis, mice were injected with Epo (300 U/kg for 5 days) and increases in hematocrits were assayed over an 11-day period. Mean ± standard error are graphed (n = 4). Note the deficit response among KitY567F/Y567F mice. (E) KitY567F/Y567F and Kit+/+ mice were dosed with both Epo (1800 U/kg) and stem cell factor (SCF) (100 µg/kg). At day 3 post dosing, bone marrow KitposCD71high erythroid progenitors were isolated and cultured in SP34-ex media. Hematopoietic cytokines were withdrawn for 4 hours, and cells were then exposed to KIT-L at 50 ng/mL. At 8 hours, cell cycle phase distributions were determined via staining with DRAQ5.

Article Snippet: In cell cycle analyses, DRAQ5 (5 µM, BOS-889-002; Biostatus, Shepshed, UK) was used together with Modfit (Verity Software, Topsham, ME, USA) as described previously [ 6 ].

Techniques: Ex Vivo, Derivative Assay, Isolation, Cell Culture, Produced, In Vivo, Injection, FACS, Flow Cytometry, Staining