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. 2022 Jun 15;23(8):e52280. doi: 10.15252/embr.202052280

Figure 2. PPARα regulates Gpx4 transcriptional activity by binding to an PPRE in Intron 3 of Gpx4, induces Gpx4 gene expression in vivo .

Figure 2

  1. Acox1 and Gpx4 mRNA was measured in liver of WT and PPARα−/− mice fed blank solvent (white bars, n = 5 mice/group) or GW7647 (black bars, n = 5 mice/group).
  2. Gpx4 protein was measured in liver of WT and PPARα−/− mice fed blank solvent or GW7647; n = 4 biological replicates.
  3. Schematic of the WT and mutant PPRE elements. The six nucleotides that were altered to form the mutant construct are underlined.
  4. A 123‐base pair fragment of intron 3 of mouse Gpx4 (position from +7679 to +7803 with respect to the transcription start site) was inserted into the pGL3 promoter vector to generate the pGL3 promoter intron 3 reporter constructs. The PPRE in intron 3 was mutated to create the mutant construct (pGL3 promoter intron 3 mut). These reporter constructs were transfected into Hep1‐6 cells. The indicated PPARα ligands were added to cell cultures 24 h before the reporter gene assay; n = 3 biological replicates. Data were calculated as the fold induction with respect to the empty vector (pGL3 promoter luciferase vector).
  5. Chromatin immunoprecipitation assays were performed on soluble formaldehyde‐crosslinked chromatin isolated from untreated and GW7647‐treated WT or PPARα−/− livers with polyclonal anti‐PPARα antibodies (anti‐PPARα) or control IgG. The final DNA extraction was polymerase chain reaction‐amplified with a primer pair that covered the sequence in intron 3 of Gpx4.

Data information: mRNA levels were normalized to 36B4 and are expressed relative to the mean value of the WT group. Data are presented as means ± SD. **P < 0.01, determined by ANOVA.