Fig. 8: Deficiency of Adam15 in macrophages and CD8+ T cells preserves activation of the mTOR-Mcl-1 prosurvival pathway when cells are exposed to CS.
a–d CD8+ T cells were isolated from the lungs of WT and Adam15−/− mice that had been exposed to CS for 3 months. a Phosphorylated S6 (p-S6-Ser235/236), total S6 (S6), and a housekeeping control (heat shock protein-90; Hsp90) were quantified in extracts of the pulmonary CD8+ T cells using Western blotting and densitometry. The p-S6-Ser235/236 signals were normalized to total S6 levels and expressed as a % of the values for WT mice. The images shown in a are representative of four mice per group. b The bars show means ± SD (n = 4 mice/group). Data were analyzed using a one-way ANOVA followed by pair-wise testing with two-tailed Students t tests. *P < 0.01 versus the WT control. c, d Intracellular protein levels of Mcl-1 and Hsp90 were quantified in pulmonary CD8+ T cells from CS-exposed mice using Western blotting and densitometry. The Mcl-1 signals were normalized to Hsp90 levels and expressed as a % of the values for WT mice. The images shown in c are representative of 3–4 mice per group. d The bars show means ± SD (n = 3–4 mice/group). Data were analyzed using a one-way ANOVA followed by pair-wise testing with two-tailed Students t tests. *P < 0.004 versus the WT control. e–g Bone marrow-derived macrophages (BMDMs) were isolated from unchallenged WT and Adam15−/− mice and exposed to 10% CSE for up to 24 h. e BMDMs were fixed, permeabilized, and immunostained with a green fluorophore for intracellular phosphorylated ribosomal protein S6 (p-S6), and the nuclei were counterstained blue with 4′,6-diamidino-2-phenylindole. Control cells were stained with a primary nonimmune rabbit (Rb) IgG. P-S6 staining in BMDMs was quantified, as described in the “Methods”. Data are mean ± SD and data were analyzed using a one-way ANOVA followed by pair-wise testing with two-tailed Students t tests. *P < 0.01 versus the control. f, g Intracellular protein levels of Mcl-1 and Hsp90 were quantified in CSE-treated BMDMs using Western blotting and densitometry. The images shown in g are representative of seven mice/group. g The Mcl-1 signals were normalized to Hsp90 levels and expressed as a % of the values for controls cells incubated without CSE. Data are mean ± SD; n = 7 mice/group. Data were analyzed using a one-way ANOVA followed by pair-wise testing with two-tailed Student’s t tests. *P ≤ 0.004 versus no CSE control belonging to the same genotype or the group indicated.
