Figure 2.
Figure 2. The apoptosis machinery remains intact in humanized Mcl-1 mice. (A) Thymocytes and splenocytes were isolated from Mcl-1hu/hu and Mcl-1wt/wt mice. Expression of MCL-1, BCL-2, BCL-XL, A1, BIM, and PUMA was detected by western blotting. Probing for HSP70 served as a loading control. Representative blots are shown. (B) Quantification of expression levels of BCL-2 family proteins in thymocytes and splenocytes from huMcl-1 (n = 9) and wild-type (n = 10) mice determined by western blotting. Chemiluminescent signals from each protein were normalized to the HSP70 loading control signal and plotted as arbitrary units; significance calculated by multiple Student t tests. (C) Lysates were prepared from thymocytes from huMcl-1 and wild-type mice and subjected to immunoprecipitation with an MCL-1 (binds both the mouse MCL-1 and huMCL-1 proteins) specific antibody. The immunoprecipitated material was immunoblotted for MCL-1 (left panel), BAK (middle panel), and BIM (right panel). Pulled-down fraction shown in top panels with whole-cell lysate (WCL) and unbound fractions shown below. (D) Thymocytes and splenic B cells were isolated from huMcl-1 and wild-type mice and exposed in culture to the indicated cytotoxic stimuli. Cell viability at 24 and 48 hours was assessed by Annexin V/PI staining and FACS analysis. (E) Thymocytes and splenic B cells were isolated from huMcl-1 and wild-type mice and treated with the MCL-1 inhibitor S63845 at the indicted concentrations. Cell viability at indicated time points was assessed by Annexin V/PI staining and FACS analysis. Data are presented as mean ± SEM; significance calculated by multiple Student t tests at each time point; *P < .05, **P < .01, ***P < .001, ***P < .0001. (F) Mice of the indicated genotypes were treated with a single dose of 150 mg/kg body weight 5-FU and survival monitored. Kaplan-Meier curves are shown; statistical analysis was performed by the log-rank (Mantel-Cox) test; *P < .05. DMSO, dimethyl sulfoxide; IP, immunoprecipitation; MW, molecular weight; ns, not significant; WB, western blot.

The apoptosis machinery remains intact in humanized Mcl-1 mice. (A) Thymocytes and splenocytes were isolated from Mcl-1hu/hu and Mcl-1wt/wt mice. Expression of MCL-1, BCL-2, BCL-XL, A1, BIM, and PUMA was detected by western blotting. Probing for HSP70 served as a loading control. Representative blots are shown. (B) Quantification of expression levels of BCL-2 family proteins in thymocytes and splenocytes from huMcl-1 (n = 9) and wild-type (n = 10) mice determined by western blotting. Chemiluminescent signals from each protein were normalized to the HSP70 loading control signal and plotted as arbitrary units; significance calculated by multiple Student t tests. (C) Lysates were prepared from thymocytes from huMcl-1 and wild-type mice and subjected to immunoprecipitation with an MCL-1 (binds both the mouse MCL-1 and huMCL-1 proteins) specific antibody. The immunoprecipitated material was immunoblotted for MCL-1 (left panel), BAK (middle panel), and BIM (right panel). Pulled-down fraction shown in top panels with whole-cell lysate (WCL) and unbound fractions shown below. (D) Thymocytes and splenic B cells were isolated from huMcl-1 and wild-type mice and exposed in culture to the indicated cytotoxic stimuli. Cell viability at 24 and 48 hours was assessed by Annexin V/PI staining and FACS analysis. (E) Thymocytes and splenic B cells were isolated from huMcl-1 and wild-type mice and treated with the MCL-1 inhibitor S63845 at the indicted concentrations. Cell viability at indicated time points was assessed by Annexin V/PI staining and FACS analysis. Data are presented as mean ± SEM; significance calculated by multiple Student t tests at each time point; *P < .05, **P < .01, ***P < .001, ***P < .0001. (F) Mice of the indicated genotypes were treated with a single dose of 150 mg/kg body weight 5-FU and survival monitored. Kaplan-Meier curves are shown; statistical analysis was performed by the log-rank (Mantel-Cox) test; *P < .05. DMSO, dimethyl sulfoxide; IP, immunoprecipitation; MW, molecular weight; ns, not significant; WB, western blot.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal