fig6

Figure 6. HepG2 cells co-cultured with Caco-2 exposed to FFAs and LPS displayed an enhanced ER and oxidative stress. The expressions of ATF4 (A), ATF6 (B), XBP1 (C), and GRP78 (D), markers of ER stress were evaluated by qRT-PCR in HepG2 cell lysates and normalized using β-actin as the housekeeping gene. Data are expressed as fold increase compared to Ctrl. ROS/RNS content was measured using a colorimetric assay (Abcam) (E). H₂O₂ levels were quantified in HepG2 lysates through DCF ROS/RNS Colorimetric Assay Kit (Abcam) (F). Malondialdehyde (MDA) was colorimetrically measured in HepG2 lysates following the manufacturer’s instructions (Abcam) (G). The number of apurinic/apyrimidinic (AP) sites was obtained by isolating total DNA from HepG2 lysates and then it was normalized to the total amount of DNA extracted (H). MnSOD2 mRNA levels were assessed by qRT-PCR and normalized to β-actin (I). For violin plots, data are expressed as median concentration (thick dashed lines) and interquartile range (dotted lines). At least three independent experiments were conducted and pooled together. ***P < 0.0001, **P < 0.001 according to one-way ANOVA. FFAs: Free fatty acids; LPS: lipopolysaccharides; ER: endoplasmic reticulum; ATF4: activating transcription factor 4; ATF6: activating transcription factor 6; XBP1: X-box binding protein 1; ROS: reactive oxygen species; RNS: reactive nitrite species; ANOVA: one-way analysis of variance.