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Blood, Vol. 111, Issue 1, 112-121, January 1, 2008
Protein kinase B (c-akt) regulates hematopoietic lineage choice decisions during myelopoiesis
Blood Buitenhuis et al.
111: 112
Supplemental materials for: Buitenhuis et al
Files in this Data Supplement:
- Figure S1. Regulation of PKB activity regulates granulopoiesis (PDF, 16 KB) -
(A) CD34+ cells were cultured in presence of (A) G-CSF to induce neutrophil differentiation during 17 days or (B) IL-5 to induce eosinophil development. Cells were cultured either in absence or presence of 10 µM LY294002 or 20 µM 1L-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) a PKB inhibitor. After 17 days of neutrophil differentiation, cytospins were made and stained with May-Grunwald Giemsa solution. Data was depicted as the percentage of progenitors, eosinophils, neutrophils and monocytes. (C/D) CD34+ cells were retrovirally transduced with myrPKB, PKBcaax or eGFP alone. Retrovirally transduced CD34+ cells were cultured in presence of (C) G-CSF to induce neutrophil differentiation or (D) IL-5 to induce eosinophil development. After 17 days of culture, transduced cells were separated from the non-transduced cells by FACS, and cytospins were made. Cytospins were stained with May-Grunwald Giemsa solution. Data was expressed as the percentage of progenitors, eosinophils, neutrophils and monocytes.
- Figure S2. Expression profiles in transduced cells (PDF, 51 KB) -
(A) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1), myrPKB (lane 2) or PKBcaax (lane 3). After 7 days of differentiation in presence of G-CSF to induce neutrophil differentiation, transduced cells were separated from the non-transduced cells by FACS, and protein lysates were made. Western Blot analysis was performed with antibodies against phosphorylated PKB (Ser473), PKB and  -actin. (B) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1) or GSK3CA (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the non-transduced cells by FACS, and protein lysates were made. Western Blot analysis was performed with antibodies against GSK-3 and Tubulin (C). CD34+ cells were retrovirally transduced with eGFP as a control (lane 1), wild C/EBP (lane 2), or C/EBPT222/226A (lane 3). After 10 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the non-transduced cells by FACS, and protein lysates were made. Western Blot analysis was performed with antibodies against phosphorylated C/EBP (T221/226), C/EBP and Tubulin.
- Figure S3. Modulation of PI3K/PKB regulates downstream effectors of PKB in granulocytic progenitors (PDF, 36 KB) -
(A) CD34+ cells were cultured in presence of G-CSF for 10 days to induce neutrophil differentiation. Cells were left untreated (lane 1) or treated with 10 µM LY294002 for 16 hours (lane 2) before protein lysates were made. Western Blot analysis was performed with antibodies against phosphorylated GSK3 (Ser9), phosphorylated S6(Ser235/236), and, as a control for equal loading, Tubulin. (B) CD34+ cells were retrovirally transduced with eGFP as a control (lane 1), or myrPKB (lane 2). After 7 days of differentiation in presence of IL-3 and IL-5 to induce eosinophil differentiation, transduced cells were separated from the non-transduced cells by FACS, and protein lysates were made. Western Blot analysis was performed with antibodies against phosphorylated PKB(Ser473), phosphorylated S6(235/236) and Tubulin.
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