Both Tnfr1 and Tnfr2 are involved in mediating the TNF-α–induced death of Tak1^−/− HSPCs. (A) Schematic diagram of the experimental design. c-kit⁺ HSPCs were isolated from BM of mice with indicated genotypes. These HSPCs were infected with MSCV-Cre-GFP virus to induce Tak1 deletion in vitro. Infected cells were cultured in HSPC medium with medium changes every day. MSCV-GFP–infected Tak1^fx/fx HSPCs were studied in parallel as controls (group 1 in B-C). (B) The percentages of infected cells (GFP⁺) from each sample were examined by flow cytometry at indicated time points and normalized to the percentage of MSCV-GFP–infected control group cells at corresponding time points. (C-D) Infected cells were purified by FACS and seeded for CFU assay. Exactly 30 000 GFP⁺ cells from each genotype were sorted and seeded into 3 plates. Numbers of colonies for each plate were counted on day 10 after cell seeding. Data shown in the figure are representative of 3 independent experiments (C). Purified infected cells with indicated genotypes were incubated in HSPC medium for 24 hours, followed by annexin V and 7-AAD staining for cell death analysis (D). **Significant reduction compared with MSCV-GFP–infected control group (P < .01). $Significant increase compared with Tak1^fx/fx group ($P < .05; $$P < .01). ##Significant increase compared with Tak1^fx/fx Tnfr2^−/− group (P < .01). &Significant increase compared with Tak1^fx/fx Tnfr1^−/− group (&P < .05; &&P < .01).