(A) Total thymocyte number in control (ctrl), Mcl-1^f/fLckCre, Bcl-2^tg, and Mcl-1^f/fLckCreBcl-2^tg mice. Control is Mcl-1^f/f or Mcl-1^+/+. (B) Representative FACS plots of control, Mcl-1^f/fLckCre, Bcl-2^tg, and Mcl-1^f/fLckCreBcl-2^tg mice. Top panel: CD4 vs. CD8 staining of total thymus. Numbers represent percent of total. Lower panel: CD44 and CD25 expression in DN cells. Numbers represent percent of DN. (C) Representative FACS plots of control (Mcl-1^f/f or Mcl-1^f/+), Mcl-1^f/fCD4Cre, Bcl-2^tg (Mcl-1^f/f or Mcl-1^f/+), Mcl-1^f/fCD4CreBcl-2^tg mice from spleen (i), thymus (ii), CD4⁺ SP gated thymocytes (iii) and CD8⁺ SP thymocytes (iv). Qa2 vs CD69 SP plots were pre-gated on TCRβ⁺. Numbers represent percent of total (i, ii) or pre-gated populations (iii, iv). (D,E) Total number of mature (TCRβ⁺Qa2⁺CD69^lo) CD4⁺ and CD8⁺ SP cells in the thymus (D) and CD4⁺ and CD8⁺ T cells (TCRβ⁺) in the spleen (E) of control, Mcl-1^f/fCD4Cre, Bcl-2^tg, Mcl-1^f/fCD4CreBcl-2^tg mice. (F,G) Percent mature (TCRβ⁺Qa2⁺CD69^lo) CD4⁺ and CD8⁺ SP cells in total thymus (F) and percent CD4⁺ and CD8⁺ T cells (TCRβ⁺) in total spleen (G) of control, Mcl-1^f/fCD4Cre, Bcl-2^tg, Mcl-1^f/fCD4CreBcl-2^tg mice. For C–G, “CD4Cre” represents Mcl-1^f/fCD4Cre. All mice were 5–8 weeks of age and data represents 2 (LckCre) or 5 (CD4Cre) separate experiments. n = 3–4 (LckCre) and 5–8 (CD4Cre), Qa2 vs CD69 not available for all experiments. For all graphs, line represents mean total/percentage. P value is illustrated as *p<0.05, **p<0.01, ***p<0.001, or not significant “ns”. Mcl-1^f/fCD4Cre and Mcl-1^f/fLckCre were significant from control by all measures displayed.

(A) Total cell numbers in thymus and spleen of Mcl-1^f/fCD4Cre mice crossed to Bak^−/− and Bax^−/−. Bars are mean + SD. Control (Ctrl) mice are Mcl-1^f/f, Mcl-1^f/fBak^+/−, Mcl-1^f/fBak^−/− or Mcl-1^f/f, Mcl-1^f/fBax^+/−, and Mcl-1^f/fBax^−/−. (B,C) Representative FACS plots for control, Mcl-1^f/fCD4Cre, Mcl-1^f/fCD4CreBak^+/−, and Mcl-1^f/fCD4CreBak^−/− (B) and Mcl-1^f/fCD4CreBax^−/− (C). Upper row shows percent T cells (TCRβ⁺) and B cells (B220⁺) in the spleen. Middle and bottom rows show percent Qa2⁺CD69^lo mature cells in CD4⁺TCRβ⁺ SP compartment (middle) and CD8⁺TCRβ⁺ SP compartment (bottom) in the thymus. (D,E) Total cell numbers of mature TCRβ⁺Qa2⁺CD69^lo SP cells in thymus (D) and CD4⁺ and CD8⁺ T cells in spleen (E) of Mcl-1^f/fCD4CreBak mice. (F,G) Total cell numbers of mature TCRβ⁺Qa2⁺CD69^lo SP cells in thymus (F) and CD4⁺ and CD8⁺ T cells in spleen (G) of Mcl-1^f/fCD4CreBax mice. Mice were 5–7 weeks of age and represent 7 (Bak) or 4 (Bax) separate experiments. n = 5–13 (Bak) or 5–9 (Bax) per group.

Anti-apoptotic proteins are displayed as diamonds, BH3-only proteins are pentagons with larger symbols representing putative “activators” and smaller symbols representing “sensitizers,” and Bak and Bax are symbolized as rectangles. (A) “Direct activation model” The anti-apoptotic proteins Mcl-1, Bcl-2 and Bcl-x_L inhibit activator BH3-only proteins Bim, Bid and possibly Puma which can directly activate Bak and Bax. Sensitizer BH3-only proteins, such as Noxa and Bad, have varying specificities and affect availability of anti-apoptotic proteins. (B) “Bak/Bax sequestration model” The anti-apoptotic proteins directly inhibit activation of Bak and Bax by binding and sequestering them from activation and/or homo-oligomerization. BH3-only proteins effect the interaction and have varying strengths due to ability to neutralize the different anti-apoptotic proteins. (C) Diagram of the major roles of the anti-apoptotic proteins throughout thymic development.

(A,B) Total thymic cellularity of control, Mcl-1^f/fLckCreBak^+/−, and Mcl-1^f/fLckCreBak^−/− (A) or control, Mcl-1^f/fLckCre, Mcl-1^f/fLckCreBax^+/−, and Mcl-1^f/fLckCreBax^−/− (B). Control = Mcl-1^f/f or Mcl-1^f/+ with Bak^+/+, Bak^+/−, or Bak^−/− (A) or Bax^+/+, Bax^+/− or Bax^−/− (B). (C) Total numbers of thymic subsets: DN, immature single positive (ISP = CD4⁻CD8⁺TCRβ⁻), DP, CD4 SP, CD8 SP (CD4⁻CD8⁺TCRβ⁺). (D,E) Representative FACS plots of control, Mcl-1^f/fLckCreBak^+/−, Mcl-1^f/fLckCreBak^−/− (D) and Mcl-1^f/fLckCreBax^−/− (E). (F,G) Mcl-1 expression in thymic subsets. Mcl-1 expression measured by the MFI ratio of anti-Mcl-1 to Rabbit IgG control. Bars are mean + standard deviation (SD). All mice were 4–7 weeks old and represent 5 separate experiments each for Bak^−/− and Bax^−/−. n = 8–11 (Bak) and 3–10 (Bax).

(A) Total cell number in thymus and spleen of Mcl-1^f/fCD4Cre mice crossed to Bim. Bars are mean + SD. Cre-negative controls are Mcl-1^+/+, Mcl-1^f/+ or Mcl-1^f/f and Bim⁺ (Bim^+/+ or Bim^+/−) or Bim^−/− as indicated. All Cre⁺ mice are Mcl-1^f/f and Bim⁺ (Bim^+/+ or Bim^+/−) or Bim^−/− as indicated. (B) Representative FACS plots of Bim^−/− control and Mcl-1^f/fCD4CreBim^−/−. Left side shows percent T cells (TCRβ⁺) and B cells (B220⁺) in the spleen. Right side shows percent Qa2⁺CD69^lo mature cells in CD4⁺TCRβ⁺ SP compartment (middle) and CD8⁺TCRβ⁺ SP compartment (far right). (C, D) Numbers of mature TCRβ⁺Qa2⁺CD69^lo SP cells in thymus (C) and CD4⁺ and CD8⁺ T cells in spleen (D) of Mcl-1^f/fCD4CreBim. Mice were 4–8 weeks of age and represent 4 separate experiments. n = 4–7.