Fig. 6

Fmo5 plays an important role in the AFLD-induced apoptosis and inflammatory response in the small intestine and liver tissues of mice and is closely related to PPARα expression and the NF-κB signaling pathway in vitro. a and b Western blot and RT-PCR assays were used to detect the protein and mRNA levels of Fmo5 and PPARα in siRNA targeting Fmo5/or PPARα transfected L02 cell or vector expressing Fmo5/or PPARα transfected L02 cells. c Co-immunoprecipitation experiments showed that Fmo5 directly interact with PPARα in L02 cell. d Double immunofluorescent staining of PPARα (red) and Fmo5 (green) showed colocalization of PPARα and Fmo5 in the L02 cells (indicated by a white arrow). e Western blot assay was used to detect the phosphorylation level of NF-κB p65 in siRNA targeting PPARα/or Fmo5 transfected L02 cell or vector expressing PPARα/or Fmo5 transfected L02 cells. f RT-PCR assay was used to detect the mRNA levels of IL-4/− 6, TNF-α, PPARα and Fmo5 in LPS treated L02 cells. g CCK-8 assay was used to detect the cell growth inhibition rate after L02 cells were treated with 0–200 nM of ethyl alcohol. h CCK-8 was used to detect the cell viability between the control and AFLD groups. AFLD cell model was shown in i. j-l ELISA assay was used to detect the levels of TG, AST, and ALT in L02 cells between the control and AFLD groups. (m) Western blot assay was used to detect the protein levels of Fmo5, PPARα, phospho-NF-κB p65 and NF-κB p65 in L02 cells between the control and AFLD groups. ELISA assay was used to detect TG, AST, ALT levels (n), IL-6, TNF-α, IL-4 levels (o) in L02 cells among the six groups. Flow cytometry assay was used to detect cell apoptosis (p) and ROS level (q) in L02 cells among the six groups. r CCK-8 assay was used to detect the viability of L02 cells among the six groups. s Western blot assay was used to detect the protein levels of cleaved caspase-3, phospho-NF-κB p65, NF-κB p65and PPARα in L02 cells among the six groups. GAPDH used as a load control. Data are presented as the mean ± standard deviation. Con+siNC vs. Con+NC = ns; AFLD+siNC vs. AFLD+NC = ns. ** P < 0.01 vs. siNC, control, Con+siNC and Con+NC groups, ## P < 0.01 vs. NC, AFLD+siNC and AFLD+NC groups