(A) The supernatant of apoptotic W3 cells was subjected to LC-MS analysis. The relative concentration of each compound is represented by the base peak intensity. (B) BMDMs were incubated with medium containing 10 μM of the indicated reagents for 1 hr, and Thbs1 mRNA levels were determined by real-time RT-PCR. (C) Apoptotic W3 cell supernatant was pretreated with 25 mU/ml apyrase at 37°C for 1 hr. BMDMs were incubated for1 hr with the pretreated or untreated supernatant, and the Thbs1 mRNA levels were determined. BMDMs were also treated with the supernatant in the presence of 10 μM AOPCP or 0.9 U/ml adenosine deaminase (ADA), and the Thbs1 mRNA levels were quantified as above. (D) BMDMs were incubated with apoptotic cell supernatant (apop) or medium supplemented with the indicated concentrations of adenosine (Ado), and the Thbs1 mRNA levels were determined. (E) The mRNA levels of the Adora1, Adora2a, Adora2b, and Adora3 expressed in BMDMs and thio-pMacs were determined by real-time RT-PCR, and normalized to β-actin mRNA. (F) BMDMs and thio-pMacs were incubated with W3 apoptotic cell supernatant and adenosine receptor antagonists, 5 nM 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (A1), 10 nM SCH58261 (A2a), 5 μM alloxazine (A2b), or 130 nM VUF5574 (A3), and Thbs1 mRNA levels were determined. (G), Thio-pMacs from Adora2a+/+ (control) or Adora2a−/− mice were incubated with medium, apoptotic or living W3 cell supernatants, and the Nr4a1 and Thbs1 mRNA levels were determined by real-time RT-PCR. Experiments were performed in triplicate, and the average values are plotted with SD (bars). All experiments were repeated at least twice with BMDM or thio-pMacs from different mice, and representative data are shown.