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. 2020 Apr 7;10:454. doi: 10.3389/fonc.2020.00454

Figure 4.

Figure 4

Determination of effects of adipocyte mediators on macrophage VEGF expression. (A) Murine 3T3-L1 were differentiated for 12 days, media was changed to fresh DMEM + insulin and cells were allowed to incubate for an additional 48 h. Media was then collected and placed onto murine J774A.1 macrophages plated 24 h prior. After 8 h of treatment with the adipocyte-conditioned media [or fresh DMEM + insulin (non-conditioned) as a control] RNA was collected, reverse transcribed and analyzed for transcriptional changes via qPCR using GAPDH and VEGFA primers (n = 3, *p < 0.05). (B) PMA-differentiated THP-1 macrophages were treated for 8 h with insulin (500 nM), leptin (1 μg/mL), or insulin (500 nM) + leptin (1 μg/mL), then RNA was collected and analyzed by qPCR (n = 4, *p < 0.05) statistical analyses were performed using standard deviation and the unpaired Student's t-test. (C) J774A.1 macrophages were treated for 8 h with insulin (500 nM), leptin (1 μg/mL), or insulin (500 nM) + leptin (1 μg/mL), then RNA was collected and analyzed by qPCR (n = 3, *p < 0.05); statistical analyses were performed using standard deviation and the unpaired Student's t-test. (D) PMA-differentiated THP-1 macrophages were treated for 8 h with IL-6 (10 ng/mL), TNF-α (10 ng/mL), or IL-6 (10 ng/mL) + TNF-α (10 ng/mL), then RNA was collected and analyzed by qPCR (n = 4, *p < 0.05); statistical analyses were performed using standard deviation and the unpaired Student's t-test. (E) J774A.1 macrophages were treated for 8 h with IL-6 (10 ng/mL), TNF-α (10 ng/mL), or IL-6 (10 ng/mL) + TNF-α (10 ng/mL), then RNA was collected and analyzed by qPCR (n = 3, *p < 0.05); statistical analyses were performed using standard deviation and the unpaired Student's t-test.