Figure 6. Akt1^only and Akt3^only macrophages are more sensitive to pro-apoptotic cells stimuli than WT and a single Akt isoform knockout cells, and Il-10 suppresses apoptosis in macrophages.

(A,B) WT, Akt2^−/−, Akt3^−/− or Akt1^only peritoneal macrophages were isolated from recipient mice (n=4/group) and two days later treated with BSA or 0.5mM PA-BSA overnight. Apoptotic cell numbers were detected by Alexa Flour 488 Annexin V/Dead cell apoptosis kit. Graphs represent data (mean ± SEM; *p<0.05 compared to WT cells treated with PA-BSA by One Way Analysis of Variance); Scale bar is 50mm.

(C-D) The percent of apoptosis in WT, Akt1^−/−, Akt2^−/− or Akt3^only macrophages after treatment with BSA or PA-BSA overnight, or incubated with human Ac-LDL (100mg/ml) plus an ACAT inhibitor Sandoz 58035 (2mg/ml) or with Ox-LDL (100mg/ml) for 48 hours. Graphs represent data (mean ± SEM) obtained from the same number (n=3/group) of mice, *p<0.05 compared to WT cells treated with PA-BSA, Ac-LDL or Ox-LDL by One Way Analysis of Variance.

(E) Akt1^only macrophages are more resistant to apoptosis than Akt3^only cells

(F) WT, Akt1^only and Akt3^only macrophages were treated with LPS (20ng/ml) for 6 hours and gene expression levels were measured by real-time PCR. Graphs represent data (mean ± SEM) obtained from the same number (n=3/group) of mice (*p<0.05 compared to WT cells treated with LPS by One Way Analysis of Variance).

(G) Macrophages were isolated from mice reconstituted with WT, Akt1^only and Akt3^only FLC and, after two days in culture, were treated with BSA or a mouse recombinant Il-10 (100ng/ml) or 0.5M PA-BSA with or without of IL-10 for 24 hours. Apoptotic cells were detected by Alexa Flour 488 Annexin V. Graphs represent data (mean ± SD) obtained from the same number (n=4/group) of mice (*p<0.05 compared to WT cells treated with PA-BSA by One Way Analysis of Variance).