Extended Data Fig. 2: Role of GAS6-MerTK signaling in ATF3 induction of macrophage.
a. Flow cytometry analysis and quantification of MerTK+ macrophages in bone marrow-derived macrophages (BMDMs) treated with rGAS6 at each time point (n = 6 per group). b. Relative mRNA expression of Mertk, Timd4, Folr2, and Cd163 in the rGAS6-treated BMDMs at different time points (n = 6 biologically independent samples per group). c. Relative mRNA expression of Atf3, Jun, and Fos in the rGAS6-treated BMDMs at different time points (n = 6 biologically independent samples per group). d. Representative IF images of ATF3 in BMDMs treated with rGAS6 or PBS for 24 h. Scale bar, 20 μm. This experiment was repeated independently triple with similar results. e. Representative IF images of MerTK (green) and ATF3 (red) in the heart of the sham operation and IR 6 h post-MI. Scale bar, 20 μm. This experiment was repeated independently triple with similar results. f. Relative mRNA expression of Atf3 in Mφ, CF, CM, and EC sorted from the heart of the sham and IR (n = 6 mice per group). g. Representative western blot analysis and quantification of the expression of phosphorylated- and total-AKT in BMDMs treated with rGAS6 for 1 h (n = 6 biologically independent samples per group). h. Relative mRNA expression of Atf3, Jun, and Fos in rGAS6-treated BMDMs transfected with scrambled control or Mertk-siRNA (n = 6 biologically independent samples per group). i. Relative mRNA expression of Atf3, Jun, and Fos in BMDMs pretreated with vehicle or LY294002 (a AKT inhibitor) (n = 6 biologically independent samples per group). j. Outline of UNC2250 (3 mg/kg) or saline treatment in WT mice subjected to IR surgery. k, l. Representative western blot analysis (k) and quantification (l) of the expression of phosphorylated- and total-MerTK, phosphorylated- and total-AKT, and ATF3 in the heart of IR mice treated with saline or UNC2250 (n = 6 mice per group). Statistical significance was evaluated using two-tailed unpaired t tests (f, g, h-Atf3, Jun, and k), unpaired Mann-Whitney U test (h-Fos), and one−way ANOVA analysis followed by Tukey’s multiple comparisons test (a–c, i). All data are presented as mean ± SEM.
