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. 2021 May 28;17(5):e1008987. doi: 10.1371/journal.pcbi.1008987

Fig 8. Mouse PER2 with C-terminal luciferase is a more potent transcriptional repressor of E-box cis-regulation in cell cultures.

Fig 8

A. Activity of the NanoLuc luciferase reporter (PE-boxNLuc) measured with the furimazine substrate in the co-transfected HEK293 cells. “Con” represents empty vectors (pCDNA3.1); B/C represents co-expression of BMAL1/CLOCK. Activity is expressed as RLU (Relative Light Units). Total amount of transfected plasmids was adjusted to the same concentration using empty vectors as needed B. Activity of the Renilla luciferase reporter (PCMV::RLuc) measured with the coelenterazine substrate in the same cell extracts as panel A. C. Activity of the NLuc luciferase reporter (PE-box) normalized by the Renilla luciferase control (PCMV). Statistical significance was analyzed by One-Way-ANOVA with Tukey’s post-hoc test at the p < 0.05 level as indicated by different letters (shared letters among groups represents no significant difference). Bars indicate Mean ±SEM; n = 4 for all groups. D. The furimazine and coelenterazine substrates do not cross react between the cells transfected with Nluc vs. Rluc. Left panel, cells that were transfected with only the PE-boxNLuc plasmid were treated separately with each of the two substrates. Right panel, cells that were transfected with only the PCMV::RLuc plasmid were treated separately with each of the two substrates. The lower readings were normalized to 1 for comparison. E. From genome sequencing of mPer2Luc mice, a C-terminal region of mouse Per2 is deleted in the mPer2Luc mice. The unexpected 72 b.p. deletion at the C-terminal end of Per2 is shown. F. Reverse transcription PCR detects a deletion of Per2 mRNA in the mPer2Luc mouse. The forward primer was designed to be in the mPer2 gene upstream of the deleted region, and the reverse primer was designed to be the 5’ end of Firefly luciferase (Fluc). The “mPer2::Luc vector” that includes the entire mPer2 gene fused to the Fluc gene without the 72 b.p. deletion was used as a positive control. Gapdh served as the control for cDNA integrity.