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. 2017 Oct 25;7:13999. doi: 10.1038/s41598-017-14376-y

Figure 7.

Figure 7

Increasing MCFA ratio protected HepG2 cells against LCFAs-induced fat accumulation, insulin resistance, autophagy impairment and lipotoxicity by downregulating Rubicon. To further confirm the involvement of Rubicon in MCFAs mediated protections, we knocked down or overexpressed Rubicon in fat-loaded HepG2 cells. The Western blotting results of CHOP and cleaved caspase 3 show that Rubicon knockdown potently attenuated LCFAs-induced lipotoxicity in HepG2 cells (A). Although Rubicon knockdown increased the SQSTM1/p62 protein level in fat-loaded cells, autophagic flux and qPCR assay results showed that Rubicon knockdown increased autophagic flux (B) and SQSTM1/p62 mRNA expression (C) simultaneously. Nile Red staining and Western blotting data also show that increasing MCFA ratio failed to rescued LCFAs-induced lipid accumulation (D), insulin resistance (E), autophagy impairment (F,G), and lipotoxicity (F) in Rubicon-overexpressed HepG2 cells, indicating that the downregulation of Rubicon is needed for MCFAs-mediated protections. For densitometric analyses of Western blotting data, β-actin was used as the loading control. Values are mean ± SEM (n = 3). * P < 0.05. n.s.: no significant difference. SDF: standard fat loading mixture (0.4 mM PA + 0.4 mM OA); MCF1: MCFA-containing fat mixture 1 (0.4 mM PA + 0.2 mM OA + 0.2 mM CA); MCF2: MCFA-containing fat mixture 2 (0.4 mM PA + 0.2 mM OA + 0.2 mM LA); Baf: Bafilomycin A1. Full-length blots are presented in Supplementary Figure S7.