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. 2017 Jun 27;58(9):1855–1868. doi: 10.1194/jlr.M077537

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Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

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Fig. 1. — Palmitate-impaired autophagic process in human hepatocellular carcinoma cells (Huh7 cells). Inhibition of autophagy in palmitate-treated cells does not aggravate the autophagic block and had no significant effect on cell death induced by palmitate. A: Huh7 cells were treated with palmitate and palmitate + 4-PBA for 24 h. MDC staining showed palmitate-treated cells with increased accumulation of autophagosomes in comparison with control. Pretreatment of 4-PBA in palmitate-treated cells reduced the accumulation of autophagosomes. Palmitate + everolimus-treated cells showed decreased autophagosomes in comparison with the only palmitate-treated cells. B: Huh7 cells were treated with palmitate and palmitate + 4-PBA for 24 h. Western blotting analysis of indicated proteins was performed and shown as an average of three independent experiments. Palmitate significantly increased the expression of mTORC1 (P < 0.001 at 0.25 mM, and 0.5 mM vs. control) and SQSTM1/p62 (P < 0.05 at 0.25 mM, and P < 0.001 at 0.5 mM vs. control) and decreased Beclin1 (P < 0.001 at 0.25 mM and 0.5 mM vs. control). Palmitate increased the LC3II/actin ratio (not significant at 0.25 mM and P < 0.001 at 0.5 mM vs. control). Pretreatment of 4-PBA in palmitate-treated cells further increased the LC3II:actin ratio (not significant at 4-PBA + 0.25 mM, and P < 0.05 at 4-PBA + 0.5 mM). The expression of Beclin1 also increased in 4-PBA + palmitate-treated cells (P < 0.05 at 4-PBA + 0.25 mM, and P < 0.01 at 4-PBA + 0.5 mM). The mTORC1 level was decreased by pretreatment of 4-PBA in palmitate-treated cells (not significant at 4-PBA + 0.25 mM and P < 0.001 at 4-PBA + 0.5 mM). SQSTM1/p62 expression was also brought down by 4-PBA pretreatment in palmitate-treated cells (not significant at 4-PBA + 0.25 mM and P < 0.01 at 4-PBA + 0.5 mM). C: Huh7 cells were treated with palmitate (0.5 mM), palmitate + 3-MA (2 mM), and palmitate + everolimus (100 nM), as was mentioned in the experimental procedures. Western blot analysis revealed that pretreatment of 3-MA in palmitate-treated cells increased the LC3II/actin in comparison with the only palmitate-treated cells. No significant effect was observed on the levels of SQSTM1/p62 and Beclin1 in palmitate + 3-MA-treated cells in comparison with the only palmitate-treated cells. Pretreatment of the autophagy inducer everolimus (100 nM) in palmitate-treated cells decreased LC3II/actin, decreased SQSTM1/p62, and increased Beclin1 in comparison with the only palmitate-treated cells. D–E: SQSTM1/p62 showed increased accumulation in palmitate-treated cells (P < 0.01, control vs. palmitate) in the presence of the autophagy inhibitors bafilomycin and CQ. Pretreatment of Baf/CQ failed to increase p62 levels in palmitate-treated cells (not significant palmitate vs. palmitate + Baf/CQ). F: Palmitate reduced the Bcl2:Bax ratio in palmitate-treated cells (0.5 mM) after 24 h (P < 0.001 control vs. palmitate). Pretreatment with 3-MA and everolimus had no further effect on the ratio (not significant palmitate vs. palmitate + 3-MA/everolimus ). G: Cellular viability was assessed by using MTT assay as mentioned in the experimental procedures. Huh7 cells were treated with palmitate (0.5 mM), palmitate + 3-MA (2 mM), and palmitate + everolimus (100 nM), as has been mentioned. Palmitate reduced the cellular viability (P < 0.001 control vs. palmitate). Pretreatment with 3-MA and everolimus had no further effect on cell viability in palmitate + 3-MA/everolimus-treated cells in comparison with the only palmitate-treated cells (P < 0.001 control vs. palmitate + 3-MA/everolimus and not significant palmitate vs. palmitate + 3-MA/everolimus). Baf, bafilomycin; Ever, everolimus; PA, palmitate; ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001: control vs. palmitate. ^#P < 0.05. ^##P < 0.01. ^###P < 0.001: palmitate vs. palmitate + 4-PBA.

Fig. 1. — Palmitate-impaired autophagic process in human hepatocellular carcinoma cells (Huh7 cells). Inhibition of autophagy in palmitate-treated cells does not aggravate the autophagic block and had no significant effect on cell death induced by palmitate. A: Huh7 cells were treated with palmitate and palmitate + 4-PBA for 24 h. MDC staining showed palmitate-treated cells with increased accumulation of autophagosomes in comparison with control. Pretreatment of 4-PBA in palmitate-treated cells reduced the accumulation of autophagosomes. Palmitate + everolimus-treated cells showed decreased autophagosomes in comparison with the only palmitate-treated cells. B: Huh7 cells were treated with palmitate and palmitate + 4-PBA for 24 h. Western blotting analysis of indicated proteins was performed and shown as an average of three independent experiments. Palmitate significantly increased the expression of mTORC1 (P < 0.001 at 0.25 mM, and 0.5 mM vs. control) and SQSTM1/p62 (P < 0.05 at 0.25 mM, and P < 0.001 at 0.5 mM vs. control) and decreased Beclin1 (P < 0.001 at 0.25 mM and 0.5 mM vs. control). Palmitate increased the LC3II/actin ratio (not significant at 0.25 mM and P < 0.001 at 0.5 mM vs. control). Pretreatment of 4-PBA in palmitate-treated cells further increased the LC3II:actin ratio (not significant at 4-PBA + 0.25 mM, and P < 0.05 at 4-PBA + 0.5 mM). The expression of Beclin1 also increased in 4-PBA + palmitate-treated cells (P < 0.05 at 4-PBA + 0.25 mM, and P < 0.01 at 4-PBA + 0.5 mM). The mTORC1 level was decreased by pretreatment of 4-PBA in palmitate-treated cells (not significant at 4-PBA + 0.25 mM and P < 0.001 at 4-PBA + 0.5 mM). SQSTM1/p62 expression was also brought down by 4-PBA pretreatment in palmitate-treated cells (not significant at 4-PBA + 0.25 mM and P < 0.01 at 4-PBA + 0.5 mM). C: Huh7 cells were treated with palmitate (0.5 mM), palmitate + 3-MA (2 mM), and palmitate + everolimus (100 nM), as was mentioned in the experimental procedures. Western blot analysis revealed that pretreatment of 3-MA in palmitate-treated cells increased the LC3II/actin in comparison with the only palmitate-treated cells. No significant effect was observed on the levels of SQSTM1/p62 and Beclin1 in palmitate + 3-MA-treated cells in comparison with the only palmitate-treated cells. Pretreatment of the autophagy inducer everolimus (100 nM) in palmitate-treated cells decreased LC3II/actin, decreased SQSTM1/p62, and increased Beclin1 in comparison with the only palmitate-treated cells. D–E: SQSTM1/p62 showed increased accumulation in palmitate-treated cells (P < 0.01, control vs. palmitate) in the presence of the autophagy inhibitors bafilomycin and CQ. Pretreatment of Baf/CQ failed to increase p62 levels in palmitate-treated cells (not significant palmitate vs. palmitate + Baf/CQ). F: Palmitate reduced the Bcl2:Bax ratio in palmitate-treated cells (0.5 mM) after 24 h (P < 0.001 control vs. palmitate). Pretreatment with 3-MA and everolimus had no further effect on the ratio (not significant palmitate vs. palmitate + 3-MA/everolimus ). G: Cellular viability was assessed by using MTT assay as mentioned in the experimental procedures. Huh7 cells were treated with palmitate (0.5 mM), palmitate + 3-MA (2 mM), and palmitate + everolimus (100 nM), as has been mentioned. Palmitate reduced the cellular viability (P < 0.001 control vs. palmitate). Pretreatment with 3-MA and everolimus had no further effect on cell viability in palmitate + 3-MA/everolimus-treated cells in comparison with the only palmitate-treated cells (P < 0.001 control vs. palmitate + 3-MA/everolimus and not significant palmitate vs. palmitate + 3-MA/everolimus). Baf, bafilomycin; Ever, everolimus; PA, palmitate; ns, not significant. *P < 0.05. **P < 0.01. ***P < 0.001: control vs. palmitate. ^#P < 0.05. ^##P < 0.01. ^###P < 0.001: palmitate vs. palmitate + 4-PBA.