Figure S1. On etoposide treatment high proportion of Jurkat cells show positivity for caspase 8 and 9.
Jurkat cells were treated with 25 mM etoposide for 8 h and stained for the caspases following instructions by the manufacturer (Calbiochem). The experiment was done in parallel with that on BMC-2 cells.
Figure S2. Many more nuclei of Xid PECs treated with LPS+IFNg show condensed morphology as compared to WT PECs. About 200 cells from triplicate cultures in each group were scanned and cells with condensed nuclei were scored as apoptotic.
Figure S3. Bone-marrow derived macrophages [BMDMs] from Xid mice are more susceptible to death than WT BMDMs on exposure to LPS+IFNg. Like Xid PECs, Xid BMDMs also produce less NO measured as nitrites as compared to WT BMDMs in response to LPS+IFNg. Data are representative on more than 7 experiments.
Methodology for the preparation of BMDMs: Mice were euthanised by cervical dislocation and cells from both femurs were harvested under sterile conditions. Cells were cultured in DMEM with 10% FCS in presence of 30% L929 culture supernatant which served as a source of M-CSF. Medium was changed every third day to remove non-adherent and/or dead cells and freshly supplemented with L-929 supernatant. On day 7 of culture cells were harvested, and stained to check their phenotype. A large proportion of cells at this time point were CD11b positive, MHC class II positive and CD86 positive as shown in panel a above. Optimum concentration of LPS and IFNg was 10 mg/ml and 30 U/ml respectively for BMDM activation. A representative experiment is shown in b. Supernatants from the wells cultured with BMDMs with and without LPS+IFNg were collected at 24 h and assayed for NO production by Greiss reaction. Data in panel c shows a representative experiment. Data represent >8 independent experiments.
Figure S4. Level of expression of CD95 is comparable between WT and Xid macrophages.
WT and Xid macrophages were stained with anti-CD95 ab coupled to PE (eBiosciences, San Diego, CA). Data are representative of 6 independent mice.
Figure S5. A schematic representation of potential signaling events contributing to macrophage death when induced by LPS+IFNg in Btk-sufficient and -deficient situations.