Fig. 2. Lyso-Gb3 induces necroptosis in an autophagy-dependent manner in ARPE-19 cells.
(A) ARPE-19 cells were exposed to the indicated concentrations of lyso-Gb3 for 24 h. Protein expressions were determined by immunoblotting with anti-RIP1, anti-RIP3, anti-MLKL, and anti-tubulin antibodies. Bar graphs show densitometric quantifications of Western blot bands. (B) ARPE-19 cells were first incubated with 3-MA (10 mM) for 1 h and then incubated with lyso-Gb3 (0.5 μM) for 24 h. Protein expression was determined by immunoblotting with anti-LC3, anti-beclin-1, anti-RIP1, anti-RIP3, anti-MLKL, and anti-tubulin antibodies. Bar graphs present densitometric quantifications of Western blot bands. (C) ARPE-19 cells were first incubated with 3-MA (10 mM) for 1 h and then incubated with lyso-Gb3 (0.5 μM) for 24 h. The cell lysates were resolved on non-reducing gel and analyzed by immunoblotting with anti-MLKL antibody. (D) The mRNA expressions of RIP1, RIP3, and MLKL were determined by quantitative real time-PCR analysis in ARPE-19 cells treated with lyso-Gb3 in the presence or absence of 3-MA (10 mM). qRT-PCR analysis was performed in triplicate. (E) ELISA assay was conducted to analyze the effect of 3-MA on IL-1β, IFN-γ, and TNF-α secretion by lyso-Gb3. ARPE-19 cells were pretreated with 3-MA (10 mM) for 1 h, followed by 24-h incubation with lyso-Gb3 (0.5 μM). (F) ARPE-19 cells were first incubated with 3-MA (10 mM) for 1 h and then incubated with TNF-α (5 ng/ml), IL-1β (10 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Protein expressions were determined by immunoblotting with anti-RIP1, anti-RIP3, anti-MLKL, and anti-tubulin antibodies. Bar graphs show densitometric quantifications of Western blot bands. The results represent three independent experiments. 3-MA, 3-methyladenine; TNF-α, tumor necrosis factor α; IL, interleukin; IFN-γ, interferon gamma. *p < 0.05 and **p < 0.01 vs. vehicle controls, #p < 0.05 and ##p < 0.01 vs. lyso-Gb3 treated cells and pro-inflammatory cytokine treated cells, one-way ANOVA followed by Bonferroni’s post-hoc test.
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