Fig 1: The effects of ECP and ECT on the expression of the PXR and AhR protein in the liver of mice exposed to eCS. Western blot analysis for the expression of (A) PXR and (B) AhR in liver tissues of mice induced by eCS. The measures represent mean ± SD. **: compared with the blank control group, p < 0.01; #: compared with the eCS model group, p < 0.05; ##: compared with the eCS model group, p < 0.01.
Fig 2: PXR expression levels and western blot bands corresponding to PXR (two bands close to 50 kDa predicted molecular weight) and histone H3 as the load control (band close to 17 kDa predicted molecular weight). Notice that in our analysis only the 50 kDa band was quantified.
Fig 3: CDK2 inhibitor dinaciclib increases the expression and activity of PXR target genes. (A) Primary mouse hepatocytes were treated with dinaciclib (4 µM) for 24 h, and the gene expression was analyzed at mRNA levels. (B) Dinaciclib promoted the oxidative activity of CYP3A4. HepG2 cells were treated with dinaciclib (0, 1, 2 or 4 µM) for 24 h, and cell lysates were prepared and assayed for CYP3A4 activity. (C,D) Dinaciclib promoted the efflux activity of P-gp. HepG2 cells were treated with or without dinaciclib (4 µM) for 24 h, then incubated with DMEM (10% FBS) supplemented with 5 µg·mL-1 Rho 123 for 30 min. The efflux activity of P-gp was analyzed immediately by flow cytometry (C) or observed under a fluorescence microscope (D). Scale bar: 50 µm. Experiments described in this figure were repeated independently at least three times, and data are expressed as mean ± SEM (n = 3). * p < 0.05 and ** p < 0.01 indicates significant difference compared with control group.
Fig 4: Inhibition of CDKs increases the protein level of PXR by suppressing its ubiquitination. (A) The cytotoxicity of dinaciclib in HepG2 cells. Hepatocytes were treated with indicated concentrations of dinaciclib for 24 h, and MTT assay was used to measure cell viability. (B–E) The effect of CDKs inhibitors on PXR protein stabilization in HepG2 cells. (B) Cells were treated with dinaciclib (4 µM) in the presence of actinomycin D (0.1 µM) with or without rifampicin (10 µM) for 24 h. (C) HepG2 cells were treated with kenpaullone (Ken, 5 µM) in the presence of actinomycin D (0.1 µM) with or without rifampicin (10 µM) for 24 h. (D) HepG2 cells were treated with kenpaullone (5 µM) or dinaciclib (4 µM) in the presence of actinomycin D (0.1 µM) for 24 h, and fluorescence intensity of PXR was detected by immunofluorescence analysis. (E) HepG2 cells were treated with PD033291 (4 µM) in the presence of actinomycin D (0.1 µM) with or without rifampicin (10 µM) for 24 h. PXR protein levels were investigated by Western blot. (F) HepG2 cells were transfected with HA-ubiquitin for 24 h and then treated with or without kenpaullone (5 µM) for 24 h. PXR protein levels were investigated by Western blot. Experiments described in this figure were repeated independently at least three times, and data are expressed as mean ± SEM (n = 3). * p < 0.05, ** p < 0.01 versus control; # p < 0.05 versus HA-ubiquitin overexpression group, ## p < 0.01 versus rifampicin-treated group.
Fig 5: CDK2-mediated phosphorylation destabilizes PXR protein and suppresses PXR-targeted genes expression. (A,B) HepG2 cells were treated with okadaic acid (0.1 µM) for 24 h, and the gene expression was analyzed at protein (A) or mRNA levels (B). Cell lysates were prepared and subjected to Western blot to determine the expression of PXR, CES1, CES2, CYP3A4 and P-gp, respectively. mRNA levels were detected by qRT-PCR. (C,D) HepG2 cells were transfected with si-CDK2 or si-CDK5 and siRNA-control for 48 h. Cell lysates were prepared and subjected to Western blot to determine the expression of PXR, CES1, CES2, CYP3A4 and P-gp, respectively. (E) The effect of CDK2 overexpression on the suppression of CES1, CES2, PXR, CYP3A4 and P-gp. Experiments described in this figure were repeated independently at least three times, and data are expressed as mean ± SEM (n = 3). * p < 0.05 indicates significant difference compared with control group.
Supplier Page from Abcam for Anti-PXR antibody