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. 2015 Jun 15;26(12):2190–2204. doi: 10.1091/mbc.E15-01-0036

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© 2015 Willy et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — CHOP directs hepatocyte secretion of TNFα and IL-8 upon exposure to palmitate. (A) RBM Myriad biomarker panel measuring the indicated secreted factors from HepG2 cells treated with either palmitate or oleate for 12 h. Values are normalized to vehicle treatment, and the number sign (^#) indicates that biomarkers were statistically significant relative to the oleate treatment group. (B, C) Sandwich ELISAs measuring IL-8 and TNFα from conditioned media of shCTRL or shCHOP HepG2 cells treated with 600 μM palmitate or oleate for 12 h or vehicle control. (D) Control (shCTRL) HepG2 cells or those knocked down for IL-8 expression (shCXCL8 #1 and shCXCL8 #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). The indicated proteins were measured by immunoblot using lysates prepared from control and shCXCL8 cells incubated for 12 h in the presence (+) or absence (–) of 600 μM palmitate (bottom). (E) Control HepG2 and those depleted for TNFα (shTNFA #1 and shTNFA #2) were treated with palmitate for 24 h, and cell death was measured by LDH release (top). Immunoblot analyses were also carried out to measure the indicated proteins in the shCTRL and shTNFA cells treated with palmitate for 12 h (+) or vehicle (–; bottom). (F, G) Sandwich ELISAs for shCXCL8 and shTNFA cells depicting repressed secreted polypeptides. (H, I) Cell migration assay for KG-1 cells using conditioned medium prepared from shCTRL, shCXCL8, or shTNFA cells treated with either vehicle or palmitate for 12 h. (J) Direct treatment indicates cell death of shCHOP cells incubated with palmitate for 24 h. Alternatively, donor shCTRL or shTNFA HepG2 cells were incubated with palmitate for 12 h. Conditioned medium was then applied to recipient shCHOP cells for 24 h, and cell death was measured by LDH release. (K) The shCHOP HepG2 cells were treated with vehicle, 600 μM palmitate, 10 ng/ml TNFα, 1000 ng/ml IL-8, or a combination of the recombinant proteins and palmitate for 24 h, as indicated. Cell death was measured by LDH release. (L) Conditioned medium was prepared from donor shCHOP HepG2 cells after 12-h treatment with vehicle or palmitate, and 10 ng/ml TNFα and/or 1000 ng/ml IL-8 was added to indicated conditioned medium before performing the KG1 cell migration assay.