Pparαb si

The ORF of T. ovatus PPARαb (ToPPARαb) (GenBank accession number: MH321826) was amplified with primers incorporating restriction sites for Nhe I and Hind III at the 5′ and 3′ ends, respectively (Supplementary Table 2). The DNA fragment was digested with the same restriction endonucleases (Nhe I and Hind III; Takara, Japan) and ligated into a correspondingly restricted pCDNA3.1-Flag vector (Invitrogen, USA). Transcription factors ToPPARαb and pGL3-basic-Elovl4a-5 of the promoter segment were chosen to determine the regulatory relationship between ToPPARαb and ToElovl4a. Detection of promoter activities were at specific time points (0 h, 3 h, 6 h, 12 h, 24 h, 48 h and 72 h). The siRNA for PPARαb (PPARαb-si) and the negative control (si-NC) were purchased from Genecreate (Wuhan, China). The PPARαb siRNA sequence is listed in Supplementary Table 2. After transfection with TOCF cells, the total protein was isolated at specific time points (0 h, 6 h, 12 h, and 24 h) as described above.

RNA interference (siRNA) of PPARαb (PPARαb-si) and corresponding negative controls (si-NC) were purchased from Genecreate (Wuhan, China). Lipofectamine RNAiMAX transfection reagent (Invitrogen, United States) was used for transfection in TOCF cells. The PPARαb siRNA sequence is listed in Supplementary Table S1. Additionally, the agonist and inhibitor of PPARα were used to clarify the role of the transcription factor in the regulation of ToElovl5 elongases. WY-14643 (0.1, 1, and 4 μmol/L, Sigma, United States) was used as a PPARαb agonist, whereas GW6471 (0.1, 1, and 4 μmol/L, Sigma, United States) was used as a PPARαb inhibitor. Total RNA was extracted from TOCF cells as described above. The experiment was performed according to Li et al. (2017) (link).