LPA converts murine monocytes into macrophages via the Akt-mTor pathway. (A) Representative phase contrast images of mouse monocytes cultured in RPMI only (control [ctrl]) or in RPMI with LPA (10 µM) or M-CSF (10 ng/mL) for up to 7 days. (B) Upper panel: Giemsa-stained images of mouse monocytes cultured in vitro in the presence of LPA or M-CSF for 7 days. Figures are representative of 10 images (n = 10). Lower panel: Immunoblot showing levels of F4/80 and b-actin in control (RPMI), LPA, and M-CSF–treated monocytes. (C) FACS plots showing proportion of CD11b⁺ and F4/80⁺ macrophages in monocytes treated with LPA or M-CSF for 5 days. (D) Quantification of percentage of CD11b⁺ and F4/80⁺ cells in monocytes cultured in medium and cells cultured in medium with LPA or M-CSF. (E-F) Quantification of CD11b- and F4/80-relative mRNA in monocytes (control) and in monocytes cultured for 5 days in medium with different doses of LPA (50 and 10 μM) or M-CSF. (G-H) Immunoblot showing levels of p-Akt, p-mTor, Akt, and mTor in macrophages differentiated from mouse monocytes cultured in medium with LPA at indicated time (t) points. (I) Representative Giemsa-stained images of LPA-derived macrophages or M-CSF–derived macrophages without or with pretreatment of Akt inhibitor (LY-294002 [Ly]). (J) FACS plots showing the proportion of CD11b⁺ and F4/80⁺ macrophages in monocytes cultured in the presence of LPA, M-CSF, or LPS and monocytes pretreated with Akt inhibitor (LY-294002) and mTor inhibitor (rapamycin) and cultured in medium containing LPA, M-CSF, or LPS, respectively. (K) Quantification of CD11b⁺ and F4/80⁺ macrophages cultured in medium with only LPA and LPA after pretreatment with Akt or mTor inhibitor, respectively. Scale bars, 100 μm. Graph presents mean ± standard deviation (SD) of 5 experiments per condition. Error bars represent SD. Student t test was used for all statistical analyses (***P < .001, **P < .01, *P < .05).