Figure 3 of Du, Mol Vis 2017; 23:185-197.


Figure 3. Activation of p38 and c-Jun N-terminal protein kinase (JNK) at least partially mediates the effects of retinol-binding protein 4 (RBP4) on cytokine production in human retinal microvascular endothelial cells (HRECs). A-C: RBP4 triggers phosphoactivation of (A) p38, (B) JNK, and (C) extracellular signal-regulated kinase (ERK) in HRECs for the indicated times from 10 min to 2 h. SFM: serum-free media only. D-F: Quantification of experiments shown in panel A-C. Phosphorylated proteins in each immunoblot are normalized to total protein content of the respective protein. Values are means ± standard error of the mean (SEM), n=3. *, p<0.05; **, p<0.01; ***p<0.001, versus SFM by one-way analysis of variance (ANOVA) with Tukey’s post hoc test. G-K: HRECs were pretreated with U0126 (ERK1/2 inhibitor, 10 μM), SB203580 (p38 inhibitor, 10 μM), and SP600125 (JNK inhibitor, 50 μM) for 2 h before incubating with RBP4 for 24 h; culture media were then collected for enzyme-linked immunosorbent assay (ELISA) -based quantification of soluble extracellular levels of (G) vascular cell adhesion molecule 1 (VCAM-1), (H) intracellular adhesion molecule 1 (ICAM-1), (I) endothelial cell selectin (E-selectin), (J) monocyte chemoattractant protein (MCP-1), and (K) interleukin 6 (IL-6). *, p<0.05; **, p<0.01; ***p<0.001, versus bovine serum albumin (BSA); #, p<0.05; ##, p<0.01; ###, p<0.001 versus RBP4 by one-way ANOVA with Tukey’s post hoc test. L: HRECs were pretreated with Cli95 (0.5 µM) for 2 h before addition of RBP4 or LPS as a positive control for 24 h. Cell lysates were analyzed by western blotting as indicated.