Figure 4.

Binding of NF‐kB to human PNPLA3 promoter mediated expression of PNPLA3 during long‐term PA treatment. A, Nucleoprotein expression trait of NF‐kB and SREBP‐1c with time during PA treatment. Nucleoprotein levels of NF‐kB and SREBP‐1c were measured at 0 h and 6 h, 12 h, and 24 h post‐treatment of PA by Western blotting and normalized to SP3; B, PA increased nucleoproteins binding to PNPLA3 promoter containing the putative NF‐kB binding site. Nucleoproteins and cytoplasmic proteins were extracted from HepG2 cells at 0 h and 12 h and 24 h post‐treatment of PA. Nucleoproteins incubated with the IRD‐700‐labelled double‐stranded DNA (dsDNA) probe of human PNPLA3 promoter containing the putative NF‐kB binding site. The binding of protein and probe was detected by EMSA. The cytoplasmic PNPLA3 protein expression was detected by Western blotting; C, BAY‐117082 attenuated the binding of nucleoprotein extracted from HepG2 cells treated with PA for 24 h and probe of human PNPLA3 promoter containing the putative NF‐kB binding site. Nucleoproteins and mRNA were extracted from HepG2 cells 24 h post‐treatment of PA with or without pretreatment of BAY‐117082. The binding of nucleoproteins and the dsDNA probe of human PNPLA3 promoter containing the putative NF‐kB binding site was detected by EMSA. PNPLA3 mRNA level was detected by real‐time PCR. Results of real‐time PCR are presented as relative mRNA levels from three independent experiments normalized to the control. #P < .05 compared with NC, *P < .05 compared with PA D, PA increased the endogenous binding of NF‐kB and PNPLA3 promoter in vivo. The endogenous binding of NF‐kB and PNPLA3 promoter was detected by CHIP‐qPCR, which was performed using an anti‐NF‐KB P65 antibody and chromatin prepared from HepG2 treated with PA for 24 h or pCMV p65 for 24 h. Fold enrichment as percentage of input DNA was calculated. Rabbit IgG was used as a mock antibody for negative control in ChIP. #P < .05 compared with control; *P < .05 compared with PA