Figure 2.

Hoxb8-withdrawal-induced cell death is completely blocked by deletion of Bax and Bak and partially blocked by Bim. (a) Hoxb8 downregulation induces Bax/Bak-dependant apoptosis. Bax^−/−;Bak^−/− Hoxb8 FDM cells were cultured with IL-3 in the presence or absence of 4-OHT. At the indicated times, viability was determined by PI exclusion and FITC-conjugated AnnexinV staining. Results are means±S.E.M. of five independent clones in two independent experiments. (b) Bim expression is repressed by Hoxb8. Western blot analysis of Bcl-2 family proteins from lysates of wild-type Hoxb8 FDM cells cultured in IL-3 after 4-OHT withdrawal and following 4-OHT re-addition on day 4 of 4-OHT deprivation. Membranes were probed with antibodies against Hoxb8, Bim, Bid, Bmf and Noxa. Arrow indicates Hoxb8. Bmf protein runs as a doublet. (c) Bim mRNA increases after Hoxb8 downregulation. Real-time PCR analysis of RNA harvested from wild-type Hoxb8 FDM cells 0, 2, 4 and 6 days after 4-OHT withdrawal. All samples were normalised against Sdh2a and Polr2a. Bim mRNA levels are expressed relative to the Bim mRNA level at 6 days after 4-OHT withdrawal (highest Bim mRNA level). Results show means±S.E.M. of three independent pools in three independent experiments. (d) Deletion of Bim reduces apoptosis after Hoxb8 downregulation. Wild-type and Bim^−/− Hoxb8 FDM cells were cultured in IL-3 in the absence of 4-OHT. Viability was determined by PI exclusion and FITC-conjugated AnnexinV staining at the indicated time points. Results show means±S.E.M. of eight independent clones in three independent experiments