Skip to main content
. 2022 May 2;132(9):e140685. doi: 10.1172/JCI140685

Figure 2. Vegfc is induced in macrophages during efferocytosis.

Figure 2

(A) Photomicrograph depicts primary BMDMs (red) cocultivated with fluorescently labeled (green) ACs. Original magnification, ×40. Bar graph shows the quantification of gene expression at the indicated time points after AC cocultivation. n = 3–5. *P < 0.03 and **P < 0.003, by 1-way ANOVA with Tukey’s test. (B) Macrophage gene expression after treatment with either ACs or LPS (100 ng/mL). n = 6 per group. **P < 0.006. (C) Representative protein immunoblots of VEGFC, 6 hours after efferocytosis and densitometric analysis. n = 6 pooled from 2 independent experiments. **P < 0.005. (D) VEGFC ELISA of macrophage supernatant 9 hours after treatment with ACs versus control. n = 6. *P < 0.01. (E) BMDMs from Cd36+/+ and Cd36–/– animals were cocultured for 3 hours with ACs. After sequential washes, cells were imaged on an Olympus fluorescence microscope, and the percentage of efferocytosis was calculated from 10 random fields per replicate. Scale bar: 20 μm. Data are representative of 2 independent experiments with n = 3 wells per group. **P < 0.01. (F) BMDMs from Cd36+/+ and Cd3–/– animals was assessed for gene expression before and after treatment with ACs. n = 3–8 per group. ***P < 0.0005 and ****P < 0.0001. (G) Macrophage Vegfc gene expression after treatment with either ACs or with ACs plus etomoxir (ETO). n = 3–4 wells per group. *P < 0.01. (H) Quantification of gene expression in macrophages treated with ACs versus ACs plus cytochalasin D (CytoD). n = 3 per group. *P < 0.02. (I) Inhibition of STAT6 with AS1517499 blocked efferocytic Vegfc induction. *P < 0.01. (J) To assess STAT6 phosphorylation, macrophages were cultured as above, and lysates were prepared in RIPA buffer and then assessed by Western blotting. n = 6 pooled samples from 2 independent experiments. *P < 0.05. Data are presented as the mean ± SEM. Statistical significance was determined by 2-way ANOVA followed by Tukey’s test (B and F–J) and 2-tailed, unpaired t test (C–E). Ctrl, control.