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. 2022 Mar 7;13(3):215. doi: 10.1038/s41419-022-04656-5

Fig. 3. ERK and PI3K/Akt signaling pathways are involved in melatonin-induced downregulation of proinflammatory factors (TNF-α, IL-8, and VEGF).

Fig. 3

A OASFs were incubated with melatonin (0–1 mM) and the Western blot assay (n = 3) determined p85, Akt, and ERK phosphorylation. B OASFs were treated with pathway inhibitors for 30 min then incubated with 1 mM of melatonin. The qPCR assay (n = 6) quantified TNF-α, IL-8, and VEGF expression. C–F OASFs were transfected with siRNAs then incubated with melatonin (1 mM) overnight. qPCR (n = 6) and ELISA (n = 6) assays measured TNF-α, IL-8, and VEGF mRNA and protein expression. G–K OASFs were pretreated with activators of ERK (C6-ceramide) 10 μM, PI3K (740-YP) 10 μM, and Akt (SC-79) 10 μM for 30 min, then incubated with melatonin (1 mM). qPCR (n = 6), ELISA (n = 6) and western blot (n = 3) assays determined the expression of TNF-α, IL-8, and VEGF, respectively. Error bars indicate means ± S.D. *p < 0.05 versus controls; #p < 0.05 versus the melatonin-treated group.