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. 2020 Apr 9;21(6):e49783. doi: 10.15252/embr.201949783

Figure 5. LAQ suppresses PRKG2 gene transcript expression via AhR negative regulation.

Figure 5

  1. The different PRKG2 promoter regions were inserted into the pGL4.1 luciferase report vector. The core promoter reporter vector of PRKG2 (−472/+131) was determined for transcription activity in cultured N2a cells, obtained from three independent experiments. One‐way ANOVA with Tukey's test; ***P < 0.001. Data are mean ± SEM.
  2. The treatment of LAQ (5 or 10 μM) caused a marked inhibition on the transcription activity of the PRKG2 promoter detected by the luciferase assay from three independent experiments. One‐way ANOVA with Tukey's test. ***P < 0.001. Data are mean ± SEM.
  3. The putative cis‐elements (in box and sequences below) in the PRKG2 promoter were determined by TFSEARCH, ConTra, and ALGGEN program analyses. Six AhR transcriptional factor binding sites were predicted according to the conserved sequence of “CACGC” or “GCGTG”.
  4. The qPCR analysis of the transcripts of Cyp1a1 and Ugt1a6a, which were mediated by AhR, in the PLP‐150Q mouse brain (corpus callosum). Note that Cyp1a1 and Ugt1a6a were significantly increased by LAQ (5 mg/kg). n = 3 mice in each group. Student's t‐test; Cyp1a1: ***P = 0.00042; Ugt1a6a; ***P = 0.00057. Data are mean ± SEM.
  5. The PRKG2 core promoter activity in N2a cells transfected with AhR siRNA or its scrambled siRNA control and then treated with 5 μM LAQ. The values of promoter activity via luciferase report assay were obtained from three independent experiments. One‐way ANOVA with Tukey's test. ***P < 0.001. Data are mean ± SEM.
  6. The semi‐PCR detection of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti‐AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8‐TCDD or DMSO for 12 h. The quantification of PRKG2 promoter DNAs associated with AhR that was immunoprecipitated by anti‐AhR in ChIP assay. The N2a cells were treated with 10 μM LAQ or 10 nM 2,3,7,8‐TCDD or DMSO for 12 h. The results were obtained from three independent experiments. One‐way ANOVA with Tukey's test. LAQ: ***P = 0.00065; 2,3,7,8‐TCDD: ***P = 0.00014. Data are mean ± SEM.

Source data are available online for this figure.