Fig. 4

Specific knock-down of PPARα transcript variants in primary human hepatocytes. a PHHs (n = 3) were transfected with siRNAs targeting PPARA-wt transcript only (siRNA-wt), PPARA-tr only (siRNA-tr), or both transcripts (siRNA-tot). Total and specific mRNA levels were determined by using specific TaqMan assays in comparison to non-targeting siRNA (siRNA-ctr; set to 1 and shown with the dotted line). Results represent means of three PHH donors with two individual replicates. Statistical significance was assessed by paired t-test in comparison to siRNA-ctr. At the bottom, PPARα protein was detected by Western blot analysis in total cell homogenates (50 μg per lane, representative western blot is shown) using a polyclonal antibody targeting the common N-terminal part of PPARα. The immunoreactive bands (upper panel) at 52 kDa (PPARα-wt) and 30 kDa (PPARα-tr) were densitometrically quantified and the intensities shown relative to the siRNA-ctr control. b Quantitative RT-PCR analysis of the selected canonical PPARα-target genes was performed in the cell lysates of A) 48 h after the transfection with the indicated siRNAs and treatment with WY14,643, in comparison to the cells transfected with siRNA-ctr and treated with the solvent control, DMSO (the dotted line) (Top panel Quantitative RT-PCR analysis of the selected proliferative genes was performed in the cell lysates of A) 48 h after the transfection with the indicated siRNAs and treatment with WY14,643 (middle panel). Quantitative RT-PCR analysis of the selected pro-inflammatory genes in the cell lysates of A) 48 h after the transfection with the indicated siRNAs and treatment with IL-6, in comparison to the cells transfected with siRNA-ctr and treated with the solvent control, PBS (the dotted line) (bottom panel). * indicates significance p < 0.05