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. 2016 Aug 23;5(9):e1183850. doi: 10.1080/2162402X.2016.1183850

Figure 5.

Figure 5.

SIRPα inhibits DC activation and survival through sequestration of p85. (A) BMDC lysates were immunoprecipitated for endogenous SIRPα and immunoprecipitates were probed with anti-SIRPα and anti-p85 antibodies. (B) PI3-Kinase activity was negatively regulated by SIRPα. BMDC lysates were collected 48 h after infection with LV-miSIRPα, LV-GFP or PBS control and assayed for PI3-Kinase activity by ELISA. (C) BMDCs were infected with adenoviral vectors (AV-GFP and AV-SIRPα) for 24 h and the level of total and phospho-Akt were ascertained by Western blotting after incubation with 100 ng/mL LPS for the indicated times. (D) Overexpression of dominant-negative Akt prevented the LV-miSIRPα-mediated IL-12 production by DCs. After infection with LV-miSIRPα for 24 h, BMDCs were infected with AV-AKT-T308A/S473A or the controls for 48 h, and the amounts of IL-12 secreted were measured by ELISA. The experiments were repeated three times yielding similar results. (E & F) Effects of inhibitors of MAPK or PI3K pathway on LV-miSIRPα-mediated IL-12 production and DC maturation. After infection with LV-miSIRPα for 24 h, BMDCs were incubated with the indicated concentrations of PI3K inhibitors or DMSO control for 24 h. ELISA measurement of IL-12 secreted by DCs and FACS analysis of surface expression of costimulatory and MHC class II molecules are shown from one of three independent experiments. *p < 0.05 vs. DMSO controls. (G) In the presence of PI3K-Akt pathway inhibitors, knockdown of SIRPα expression failed to enhance DC survival. After infection with LV-miSIRPα or incubation with 100ng/mL LPS for 24 h, BMDCs were treated with the indicated concentrations of PI3K inhibitor Ly294002, Wortmannin or DMSO control. Cell viability was assessed 2 d later using PI staining. Experiments were repeated three times yielding similar results. (H) LV-miSIRPα-transduced BMDCs were further infected with adenoviral vectors expressing dominant-negative mutant of Akt (AV-AKT-T308A/S473A) or GFP control. The survival of DCs was examined as described above. *p < 0.05 versus GFP and PBS controls. Experiments were repeated three times with similar results.