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Fig. S1. Rapid induction of neutrophil apoptosis by Rroscovitine. a, Morphology of neutrophils cultured with R-roscovitine. Neutrophils were incubated in DMSO (control) or R-roscovitine (30 μm) for 6 h. Photomicrographs (original magnification, ×400) of cytospins. Examples of apoptotic neutrophils are indicated with solid arrows. b, Neutrophils were incubated in GM-CSF (10 ng/mL), R-roscovitine (1– 30 μm) or DMSO (vehicle control). Neutrophil apoptosis was assessed morphologically. Data represent the mean± SEM of four independent experiments, each performed in triplicate (*P<0.05 compared with DMSO values using ANOVA). c, Effect of R-roscovitine on neutrophil apoptosis (FACS analysis). Neutrophils were incubated with R-roscovitine (30 μm) or TNF-α (100 U/mL) for 6 h. Apoptosis was assessed by flow cytometry using FITClabeled human Annexin-V/PI. Dotplots representing fluorescence are from one experiment representative of three others.

Fig. S2. R-roscovitine induces rapid apoptosis of human eosinophils, not primary necrosis. Eosinophils (5×106/mL) were incubated with DMSO (control), dexamethasone (1000 nm), R-roscovitine (30 μm) or ketotifen fumarate (1 mm). Dotplots representing annexin-V and PI fluorescence of eosinophils assessed at 24 h. Data are from one experiment representative of two others.

Fig. S3. Modulation of apoptosis by CDK inhibitors. Eosinophils (5×106/mL) were incubated with DMSO (control), R-roscovitine (ROS) (30 μm), alsterpaullone (ALS) (1– 10 μm), purvalanol A (PUR) (1– 30 μm) or CDK1/2 inhibitor III (100– 300 nm) for 6 h and 24 h. Eosinophil apoptosis was assessed morphologically. Data represent the mean± SEM of three independent experiments (*P<0.05 compared with control values using ANOVA).

Fig. S4. R-roscovitine reduces Mcl-1L expression in human eosinophils. Highly purified eosinophils were isolated using Robosep® and incubated with DMSO (CTL) or R-roscovitine (30 μm) for 4 h before cell lysates (30 μg/lane) were obtained as described in ‘Materials and Methods’. 5 μg of BJAB whole cell lysate and 30 μg of fresh neutrophil lysate were used as a positive control. Blots are representative of at least two independent experiments.

Fig. S5. Morphological features of human eosinophil apoptosis and secondary necrosis induced by R-roscovitine. Eosinophils were incubated with DMSO (control) or R-roscovitine (18 μm). Eosinophils were harvested at 24 h and morphology assessed by TEM (original magnification, ×2950). a, The majority of eosinophils incubated with DMSO (control) show a healthy appearance with well-preserved membrane integrity and bilobed nuclei (solid arrow) but apoptotic eosinophils also occur with rounded-up nuclei and condensed chromatin (open arrow). b, Eosinophils incubated with R-roscovitine display a heterogeneous morphology, including apoptotic cells. c, Eosinophils also exhibit late apoptotic morphology with vacuolated cytoplasm and intact cell membrane (open arrowhead). d, Necrotic anucleate eosinophils (closed arrowhead) were also present in R-roscovitine samples.

Fig. S6. R-roscovitine does not induce eosinophil degranulation or cytolysis. a, Effect of R-roscovitine on LDH release by granulocytes. Neutrophils or eosinophils were incubated with GM-CSF or IL-5 (10 ng/mL), R-roscovitine (1– 30 μm) or with DMSO. LDH release was measured after 24 h. 100% LDH release was defined using the lysis solution provided in the manufacturer' s kit. Neutrophil data represent the mean± SEM of four independent experiments, each performed in triplicate and eosinophil data represent the mean± SEM of three independent experiments, each performed in triplicate (*P<0.05 compared with DMSO values using ANOVA). b, Effect of R-roscovitine on constitutive and IL-5- induced EDN release from eosinophils. Eosinophils were incubated with GM-CSF (10 ng/mL), with R-roscovitine (1– 30 μm) or with DMSO. Data represent the mean± SEM of five independent experiments (*P<0.05 compared with control values and **P<0.05 compared with IL-5 values using a Mann-Whitney test).

Fig. S7. Induction of apoptosis in bone marrow-derived neutrophils and eosinophils by R-roscovitine. a, Bone marrow-derived granulocytes (1×106/mL) were incubated in RPMI 1640 with DMSO (control) or with R-roscovitine (10 μm and 30 μm). Cells were harvested at 8 h and morphology assessed by light microscopy. Data are from a single experiment. b, Morphology of granulocytes cultured with R-roscovitine. Photomicrographs (original magnification, ×600) of bone-marrow derived cell cytospin preparations at 8 h. Cells incubated with DMSO (control) display normal morphology and granulocytes incubated with 30 μm R-roscovitine display increased apoptotic morphology. Examples of non-apoptotic granulocytes are marked with an open arrowhead (neutrophils) and open arrow (eosinophils), whereas apoptotic granulocytes are marked with a solid arrowhead (neutrophils) and marked with a solid arrow (eosinophils).

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