|
|
Blood, Vol. 109, Issue 4, 1678-1686, February 15, 2007
Constitutive activation of Stat5 promotes its cytoplasmic localization and association with PI3-kinase in myeloid leukemias
Blood Harir et al.
109: 1678
Supplemental materials for: Harir et al, Vol 109, Issue 4, 1678-1686
Files in this Data Supplement:
- Figure S1. Transduction of TAT-Akt fusion proteins into Ba/F3 cells expressing the constitutively active Stat5A1*6 mutant (JPG, 39.2 KB)
-
(A) TAT-wtAkt and TAT-dnAkt recombinant proteins were purified from bacterial lysates by affinity chromatography on IMAC columns. The purity of the 2 first eluates (E1 and E2) obtained for TAT-wtAkt (lanes 1 and 3) or TAT-dnAkt (lanes 2 and 4) proteins was assessed by Coomassie blue–stained SDS-PAGE. Both purified proteins migrated with an expected molecular weight of 62 kDa. The quantity of purified proteins was estimated from the gel using standard concentrations of BSA. For most of the purifications, the protein concentration obtained was approximately 1 µM. (B) Ba/F3 cells expressing Stat5A1*6 were incubated with TAT-wtAkt and TAT-dnAKT proteins for the indicated times. Cells were extensively washed (6 times), and extracts were prepared to detect the presence of TAT-Akt proteins by Western blotting using anti–Pser473-Akt and Akt antibodies. TAT-wtAkt and TAT-dnAkt were detected in the Stat5A1*6-expressing cells within 30 minutes of incubation time and their expression remained stable for at least 48 hours. Moreover, we also found that both fusion proteins were phosphorylated on the residue ser473, indicating that TAT-Akt and TAT-dnAkt were activated as was endogenous Akt in Stat5A1*6-expressing Ba/F3 cells.
- Figure S2. Biologic activity of the TAT-Akt fusion proteins in Stat5A1*6-expressing Ba/F3 cells (JPG, 52.7 KB)
-
(A) Cells were incubated with increasing concentrations of TAT-wtAkt and TAT-dnAkt proteins or vehicle (PBS) as indicated for 24 hours, and cell growth was determined using the trypan blue dye exclusion method. The growth of Stat5A1*6-expressing cells was clearly inhibited in a dose-dependent manner with the TAT-dnAkt, whereas it was unaffected with the TAT-wtAkt. The maximum inhibitory effect was observed with 100 nM TAT-dnAkt. (B) Cells were grown in the presence or not (PBS) of TAT-wtAkt or TAT-dnAkt fusion proteins (100 nM), and the cells were counted daily using the trypan blue dye assay. The use of such concentration of TAT-dnAkt proteins induced cell-growth inhibition as detected after 12 hours of culture. In contrast, transduction of TAT-wtAkt proteins at the same concentration did not modify the proliferation of Stat5A1*6-expressing cells. (C) The percentage of living cells transduced with the TAT-dnAkt rapidly decreased as a function of time, indicating that the survival of the cells was affected by this fusion protein. To analyze the effect of the TAT-dnAkt recombinant protein on apoptosis, cells expressing Stat5A1*6 were transduced or not (PBS) with the TAT-Akt fusion proteins or incubated or not (EtOH) with LY294002 during 24 hours and then labeled with propidium iodide. DNA content was next evaluated by flow cytometry. Results showed that transduction of TAT-dnAkt was efficient as LY294002 to induce the appearance of a sub-G1 cell population, whereas transduction of TAT-wtAkt was without effect, indicating that cells transduced with the TAT-dnAkt protein underwent apoptosis.
- Figure S3. Transduction of TAT-dnAkt induces Forkhead transcriptional activity in Ba/F3 cells transformed by Stat5A1*6 (JPG, 45.2 KB)
-
(A) IL-3–starved Ba/F3 cells were incubated with TAT-wtAkt and TAT-dnAkt proteins for 12 hours and then stimulated with IL-3 for 30 minutes. Cell extracts were prepared and analyzed by Western blotting with anti–phospho-FKHR and FKHR antibodies. Transduction of TAT-dnAkt resulted in a dramatic inhibition of FKHR phosphorylation. (B) Cell extracts from Stat5A1*6-expressing Ba/F3 cells transduced with TAT-Akt recombinant proteins were analyzed by Western blotting with anti-p27kip1 and anti-Bim antisera (left). A similar experiment was performed with Stat5A1*6-expressing and parental Ba/F3 cells incubated with LY294002 (10 µM; right). Extracts from Ba/F3 cells deprived of IL-3 during 16 hours were also loaded as control. Bim and p27kip1 protein expression was up-regulated in Stat51*6-expressing cells transduced with TAT-dnAKT but not with TAT-wtAkt (left). Similar results were obtained with cells treated with LY294002 (right).
- Figure S4. Tyrosine phosphorylation and cytoplasmic localization of tetramer-deficient cS5F mutants in Ba/F3 cells (JPG, 45.1 KB)
-
(A) cS5F and the tetramer-deficient mutants cS5FW37A and cS5F136 cDNAs were cloned into the bicistronic expression vector pIREShrGFP carrying a Flag tag sequence. The resulting constructs and the empty vector (mock) were introduced in Ba/F3 cells by electroporation, and, the following day, GFP+ cells were sorted by flow cytometry and were deprived of IL-3. Sixteen hours later, expression of the Flag-tagged cS5F proteins in transfected cells were analyzed by Western blot using anti–P-Y-Stat5 and anti-Flag antibodies. (B) Immunocytochemical detection of phosphorylated cS5F and the tetramer-deficient cS5F mutants in transfected Ba/F3 cells. In each case, cells were spun on cytospin slides and analyzed by indirect immunocytochemistry using an anti–P-Y-Stat5 antibody (AX1).
- Figure S5. Tyrosine phosphorylation of wt-Stat5 does not occur in cS5F-expressing cells (JPG, 45.7 KB)
-
(A) Sorted GFP+ BM cells from cS5F-grafted mice (cS5F BM) or from control GFPv mice (GFPv BM) were cultured in the presence of SCF (10 ng/mL) (cS5F BM) or in the presence of SCF and IL-3 (10 ng/mL). GFPv BM were then starved of IL-3 for 4 hours and then stimulated with IL-3 for 30 minutes. Extracts from GFPv BM cells and cS5F BM cells stimulated or not with IL-3 were analyzed by Western blot with the indicated antibodies. (B) Stat5 was also immunoprecipitated from these extracts, and the presence of P-Y-Stat5 in the immunoprecipitates was detected by Western blot analysis with the indicated antibodies. Cell lysates were also immunoprecipitated with an isotypic control IgG antibody. (C) The bicistronic expression vector pIREShrGFP carrying cS5F fused to a Flag tag or the empty vector were introduced in Ba/F3 cells by electroporation. The following day, GFP+ cells were sorted by low cytometry and deprived of IL-3. Sixteen hours later, extracts from Flag-tagged cS5F-transfected cells or control cells (vector) stimulated or not with IL-3 were prepared and the presence of P-Y-Stat5 was detected by Western blot analysis. Membranes were also probed with anti-Stat5 or anti-Flag antibodies.
- Figure S6. Transient expression of cS5F induces activation of the PI 3-kinase/Akt pathway in Ba/F3 cells (JPG, 38 KB)
-
(A) Ba/F3 cells were electroporated with the bicistronic cS5F expression vector or the empty vector. The following day, GFP+ cells were sorted by flow cytometry and grown in the absence or presence of IL-3. Living cells were daily enumerated using the trypan blue dye exclusion method. Results shown are representative of 3 different transfection assays. (B) Sorted GFP+ cells expressing cS5F grown in the absence of IL-3 were treated or not with the PI3-kinase inhibitor LY294002 (10 µM). The number of living cells was daily determined using the trypan blue dye exclusion method. Results shown are representative of 3 different transfection assays. (C) Extracts from cells transfected with the bicistronic Flag-tagged cS5F expression vector or the empty vector were analyzed by Western blot with the indicated antibodies.
|
|
