FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells -- Pellegrini et al. 106 (5): 1581 Data Supplement - Supplemental Figures for: Pellegrini et al, Vol 106, Issue 5, 1581-1589 -- Blood

FADD and caspase-8 are required for cytokine-induced proliferation of hemopoietic progenitor cells
Blood Pellegrini et al. 106: 1581

Supplemental Figures for: Pellegrini et al, Vol 106, Issue 5, 1581-1589

Files in this Data Supplement:

Figure S1. Generation and analysis of VavP-FLAG-CrmA transgenic mice (PDF, 884 KB) - (A) Tail DNA was extracted from primary and first generation VavP-FLAG-CrmA transgenic mice. Dot hybridization to a ³²P-labeled SV40 probe was used to detect the presence of the transgene. DNA was spotted in duplicate, and the transgene copy number can be estimated by comparing the intensity of hybridization to that of control SV40 DNA of known concentration. These are typical results, with 2 examples highlighting a transgene positive (solid line) and a negative (broken line) mouse. (B) PCR using SV40-specific primers was used to detect transgene sequences in primary transgenic mice or first-generation descendants. Results are representative of multiple screens. (C) Western blots on extracts of 106 cells from spleen or thymus of VavP-FLAG-CrmA transgene-positive mice were probed with anti-FLAG mAb. (D) In an alternative method for detecting transgene expression, cells from the lymph nodes (LN), thymus, or bone marrow of VavP-FLAG-CrmA transgenic mice were stained with antibodies to Thy1, CD8, CD4, Gr-1, and/or Mac-1; sorted in a FACS machine; fixed; permeabilized; stained with anti-FLAG mAb; and analyzed in a FACScan. Intracellular staining of thymocytes from lck-FLAG-FADD-DN transgenic mice with anti-FLAG mAb was the positive control. Results are representative of multiple screens (>12 PCR-positive mice). (E) Granulocytes (Mac-1⁺Gr-1⁺) were FACS-sorted from the bone marrow of vavP-FLAG-CrmA transgene positive progeny and control mice and were cultured for 24 hours in medium alone or in the presence of oligomerized recombinant FasL (100 ng/mL). Cell viability was determined by staining with propidium iodide (PI) and FACS analysis. Results shown are representative of more than 12 assays.
Figure S2. Apoptosis assays with hemopoietic cells from VavP-FLAG-FADD-DN transgenic mice (PDF, 878 KB) - Immature thymocytes (CD4⁺CD8⁺), monocytes (Mac-1⁺Gr-1^–), mature T-cells (Thy1⁺), and B cells (B220⁺) were sorted by flow cytometry from the thymus, bone marrow, or lymph nodes of VavP-FLAG-FADD-DN transgene-positive and control mice and were cultured for 24 hours in medium alone (control) or in the presence of oligomerized FasL. B cells and monocytes were activated in vitro with LPS plus IL-2, IL-4, and IL-5 or with IFNγ, respectively, prior to apoptosis assays. Cell viability was assessed by PI staining and FACS analysis. Results are representative of more than 12 analyses.
Figure S3. Apoptosis assays with hemopoietic cells from VavP-FLAG-CrmA transgenic mice (PDF, 577 KB) - Immature thymocytes (⁺CD8⁺), monocytes (Gr-1^–Mac⁺), and B cells (B220⁺) were FACS-sorted from the thymus, bone marrow or lymph nodes of VavP-FLAG-CrmA transgene-positive and control mice and were cultured for 24 hours in medium alone (control) or in the presence of oligomerized recombinant FasL (100 ng/mL). B cells and monocytes were activated in vitro prior to death assays. Cell viability was assessed by PI staining and FACS analysis and was compared with that of WT cells. Results are representative of more than 12 analyses.
Figure S4. Jurkat cells infected with retroviruses produce functional proteins (PDF, 382 KB) - (A) Jurkat cells expressing the murine ecotropic retrovirus receptor were infected with retroviruses encoding FLAG-CrmA; mutant FLAG-CrmA, which does not inhibit caspase-8; FLAG-caspase-8-DN; FLAG-caspase-9-DN; FLAG-FADD-DN; mutant FLAG-FADD-DN; or the cytoplasmic region of Fas (FLAG-cFas). All constructs contained an internal ribosome entry site (IRES) plus green fluorescent protein (GFP). Infected Jurkat cells were sorted by FACS for GFP expression and then lysed for Western blot analysis. Western blots prepared by SDS-PAGE of lysates from 10⁶ infected (GFP⁺) cells were probed with anti-FLAG mAb to detect FLAG-tagged proteins. Probing with anti-HSP70 antibody was used as a loading control. (B) FACS-sorted retrovirally infected (GFP⁺) Jurkat cells were treated with oligomerized recombinant FasL or etoposide, and cell viability was assessed at various time points by PI staining and FACS analysis. Data points represent mean ± SD of 3 independent experiments.