Fig. 3. Conditioned media of lyso-Gb3 treated ARPE-19 cells increases endothelial necroptosis and inflammation.
(A) Schematic view of the experiment design. (B–D) ARPE-19 cells were pretreated with necrostatin (30 μM), GSK’872 (30 μM), or 3-MA (10 mM) for 1 h and then incubated with lyso-Gb3 (0.5 μM) for 3 h. Media were then replaced with fresh medium. After overnight (O/N) incubation, the conditioned medium (CM) was recovered and then transferred to HUVECs for further incubation for 24 h. ARPE-19 cells and HUVECs were immunoblotted using anti-RIP1, anti-RIP3, anti-MLKL, anti-LC3, anti-beclin-1, anti-VCAM-1, anti-ICAM-1, and anti-tubulin antibodies. Bar graphs show densitometric quantifications of Western blot bands. 3-MA, 3-methyladenine; HUVEC, human umbilical vein endothelial cell. *p < 0.05 and **p < 0.01 vs. vehicle controls, #p < 0.05 and ##p < 0.01 vs. lyso-Gb3 treated cells, one-way ANOVA followed by Bonferroni’s post -hoc test.
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