Supplementary Figure 1 Caspase-8 deficient platelets respond normally to agonists and ABT-737 (A) Surface expression of GPIX, GPIb, GPVI and CD41

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1 Supplementary Figure 1 Caspase-8 deficient platelets respond normally to agonists and ABT-737 (A) Surface expression of GPIX, GPIb, GPVI and CD41 were assessed on purified platelets from Casp8 fl/fl and Casp8 Pf4 /Pf4 mice by flow cytometry. (B and C) Platelets were treated with the single agonists ADP ( µm), Convulxin ( ng ml -1 ), PAR4-AP ( µm) or were left untreated for 20 min at room temperature, then (B) P-selectin exposure, or (C) activated CD41/CD61 integrin (JON/A) was assessed by flow cytometry. (D) Platelet phosphatidylserine exposure determined by flow cytometric analysis of Annexin V staining. Platelets were treated with the dual agonists thrombin plus convulxin (Thr U ml -1 ; Cvx ng ml -1 ) or ionophore A23187 (1 µm) for 10 min at room temperature, or with the intrinsic apoptosis pathway inducer ABT-737 ( µm) for 90 min at 37 C. n=3-7 mice per genotype for each treatment. Data represent mean s.d. 1

2 Supplementary Figure 2 FasL triggers apoptosis in megakaryocytes Representative still images from time-lapse video microscopy of cultured BM derived BSA-gradient purified megakaryocytes treated with cross-linked FasL 1 µg ml -1. Control indicates megakaryocytes treated with FLAG mab M2 displaying proplatelets. Cell permeable Mitotracker Red FM stains mitochondria (red) and was used as a viability stain. Note decreased red intensity as FasL treated megakaryocytes die and become positive for the nuclear stain DAPI (blue). FasL treated cells #1 and #2 appear viable at time 0. Blebbing occurs at h. At 9 h #1 collapses, becomes permeable (DAPI positive) as indicated by yellow arrow and cell material extends out from the cell indicted by dotted white lines. Cell #2 contracts at 9h and later becomes DAPI positive (data not shown). Mitotracker Red FM, DAPI and cross-linked FasL were added 1h before commencing time-lapse images. Videos start at 8.5 h (Control ) and 1.5 h (FasL). Frames were captured every 10 min for 21 h. Time (h). Bar, 50 μm. 2

3 Supplementary Figure 3 Expression of Caspase-8 in platelets from Fas lpr/lpr BM chimeras Western blot analysis of platelet protein lysates from chimeric mice 8 weeks after reconstitution with Casp8 Pf4 /Pf4 bone marrow (Casp8 Pf4 /Pf4 Fas lpr/lpr ). The arrows point to Caspase-8. Each lane represents an individual mouse. # indicates mice excluded from the study. Probing for actin was used as control for protein loading. WT=Wild-type. 3

4 Supplementary Figure 4 Blood counts in Fas lpr/lpr BM chimeras treated with FasL Fas lpr/lpr mice reconstituted with wild-type CD45.1 GFP + BM (Wild-type Fas lpr/lpr ), Fas lpr/lpr mice reconstituted with Casp8 Pf4 /Pf4 BM (Casp8 Pf4 /Pf4 Fas lpr/lpr ), and Wildtype mice were injected i.v. with 0.25 µg g -1 body weight of recombinant FasL aggregated with 2 µg FLAG (M2) mab per µg FasL or, as a negative control, with FLAG mab M2 alone. Wild-type mice were culled at 2.5 h and BM reconstituted Fas lpr/lpr mice at 24 h post injection. Data represent mean s.d. n=5-6 Wild-type Fas lpr/lpr ; n=6 Casp8 Pf4 /Pf4 Fas lpr/lpr and n=2 Wild-type mice per group. Student s unpaired t-test, **P <

5 Supplementary Figure 5 Analysis of mice lacking Bak, Bax and Caspase-8 in the megakaryocytic lineage (A) Efficient deletion of BAK, BAX and Caspase-8 in platelets from Bak -/- Bax Pf4 /Pf4 Casp8 Pf4 /Pf4 mice. Western blot analysis of platelet lysates from 2 Wild-type (control) and 2 Bak -/- Bax Pf4 /Pf4 Casp8 Pf4 /Pf4 mice. / indicates Pf4 Cre-specific deletion. Probing for actin was used as a control for protein loading. (B) Ploidy distribution profile of CD41 + bone marrow cells in Wild-type, Bak -/- and Bak -/- Bax Pf4 /Pf4 Casp8 Pf4 /Pf4 mice, as determined by flow cytometry. n=4 mice per genotype. (C) Serum thrombopoietin levels. n=8 Casp8 fl/fl, n=14 Casp8 Pf4 /Pf4 and n=12 Bak -/- Bax Pf4 /Pf4 Casp8 Pf4 /Pf week old mice. Data represent mean s.d. Student s unpaired t-test, *P < 0.05; **P < 0.005; ***P <

6 Supplementary Figure 6 Uncropped western blots (A) Western blot included in Fig. 2A. (B) Western blot included in Fig. 3A. (C) Western blots included in Fig. 4A. The lower band in the second panel is actin. (D) Western blots included in Supplementary Fig. 3. 6

7 Genotype Treatment MKs 0h 24h 48h 72h Wild-type Control Total 135 ± ± ± ± 23 PPF 0 16 ± 5 30 ± ± 9 FasL Total 135 ± ± ± 26 9 ± 7 PPF 0 4 ± 2 2 ± 2 1 ± 1 Casp8 Pf4 /Pf4 Control Total ± 6 34 ± 5 28 ± 6 PPF 0 12 ± 4 18 ± 8 11 ± 1 FasL Total ± 8 35 ± 5 31 ± 5 PPF 0 14 ± 5 17 ± 3 11 ± 3 Fas lpr/lpr Control Total ± 7 31 ± 2 23 ± 2 PPF 0 18 ± 7 14 ± 2 7 ± 3 FasL Total ± 3 33 ± 4 24 ± 3 PPF 0 18 ± 3 16 ± 2 9 ± 3 Bak -/- Bax -/- Control Total ± ± ± 1 PPF 0 24 ± 4 57 ± ± 1 FasL Total ± 6 56 ± ± 8 PPF 0 7 ± 4 8 ± 4 1 ± 1 Supplementary Table 1 FasL treatment leads to failure of proplatelet formation E13.5 foetal liver cells were cultured in serum free medium in thrombopoietin for 3 days. On day 4, large megakaryocytes (MKs) were purified from a BSA gradient and reseeded (time 0) to investigate proplatelet formation after treatment with crosslinked FasL 2 µg ml -1. Control indicates MKs treated with FLAG mab M2 only. PPF=proplatelet forming MKs. Total MKs include PPF MKs. n=8 Wild-type; n=4 Fas lpr/lpr ; n=4 Casp8 Pf4 /Pf4 ; n=2 Bak -/- Bax -/- biological replicates. Data are absolute numbers presented as mean s.d. 7