Fig 2: Poly(ADP-ribosyl)ated PXR promotes its activation and recruitment to CYP3A11 gene.a Relative CYP3A11 or mutant CYP3A11 (mut-PXRE) promoter luciferase reporter activity in primary mice hepatocytes treated with PJ34(15 µM) or APAP(300 µM) for 24h. b Relative CYP3A11 or mutant CYP3A11 (mut-PXRE) promoter luciferase reporter activity in primary WT or PARP1-/- mice hepatocytes with or without APAP treatment (300µM; 12 h). c HepG2 cells were transfected with the GAL4 reporter plasmid together with Gal4-PXR-LBD or Gal4-PXR-LBD mutant, and then exposed to activated PARP1 or inactivated PARP1 (mut-PARP1) for 24 h. Luciferase activity was assayed, and normalized. The data were represented by mean ± SEM of four independent experiments. *P < 0.01 vs. APAP. d CHIP-PCR assays using an anti-PXR antibody for amplification of CYP3A11 promoters in primary mice hepatocytes treated as indicated. e CHIP-PCR assays using an anti-PXR antibody for amplification of CYP3A11 promoters in primary WT or PARP1-/- mice hepatocytes. f In re-CHIP assays, chromatin was first immunoprecipitated with an anti-PXR antibody and was then re-immunoprecipitated with an anti-PAR antibody, an anti-PARP1 antibody, IgG, or an anti-RNA Pol II antibody. IgG served as a negative control

Fig 3: PXR protected barrier function from LPS damage. a TEER changes in Caco-2 cell monolayers treated with LPS for 24 or 48 h. b and c Western blot analysis of claudin-1, occludin, and ZO-1 protein levels in Caco-2 cells treated with LPS (n = 4 per group, representative data from three independent experiments). d Western blot analysis of PXR in Caco-2 treated with LPS. e Confirmation of PXR overexpression by Western blot. f and g Western blot analysis of the components of tight junctions (Claudin-1, Occludin, and ZO-1) in Caco-2 cells treated with LPS for 48 h (n = 4 per group, representative data from three independent experiments). h FD4 flux of basolateral medium was measured after Caco-2 cells were treated with LPS or not treated for 48 h (n = 5 per group, representative data from four independent experiments). Data are expressed as mean values ± SD. Statistical analyses were performed with Student’s two-tailed unpaired t-test in (c and g), *p < 0.05; **p < 0.01; n.s., not significant; TEER, transepithelial electrical resistance; FD4, FITC-dextran 4 kDa

Fig 4: Levels of PXR (endogenous or ectopically expressed) in colon cancer cell models and human hepatocytes. (a) Western blot analysis of hPXR (detected by anti-PXR) and p53 (detected by anti-p53) in HCT116 (p53+/+, p53-/-) cells, LS180 (p53+/+) cells, and RKO (p53+/+, p53-/-) isogenic pairs stably transduced with either lentiviral FLAG-tagged EV or FLAG-tagged hPXR (PXR). ß-Actin (detected by anti-ß-actin) was used as a loading control. (b) Average Ct values from quantitative real-time PCR analysis of hPXR (NR1I2) mRNA expression in the RKO isogenic pair (p53+/+ (RKO p53+/+), p53-/- (RKO p53-/-)), the HCT116 isogenic pair (p53+/+ (HCT116 p53+/+), p53-/- (HCT116 p53-/-)), and LS180 (LS180 p53+/+) cells stably transduced with EV or PXR and primary human hepatocytes.

Fig 5: The PXR-ATF3 interaction is required for ATF3 regulation. (A) Identification of ATF3 domains for PXR binding. 1 µg ATF3 truncated proteins fused to GST were immobilized onto glutathione agarose beads and incubated with 5 µl in vitro translated PXR for GST-pulldown assays. The pulled-down proteins were detected by Western blotting using the PXR antibody. (B) The ATF3 K42R mutation significantly reduced the binding between ATF3 and PXR. H1299 cells were transfected with 1.5 µg FLAG-ATF3 wild type or mutants, and 1.5 µg PXR as indicated for 2 days. FLAG-immunoprecipitation (IP) assays were performed using FLAG antibody to precipitate. (C) The ATF3 K42 was essential for ubiquitination of ATF3 regulated by PXR. H1299 cells were transfected with 1 µg FLAGATF3 wild type or mutants, and 2µg HA-ubiquitin, and/or 4 µg PXR as indicated for 2 days, and then subjected to FLAG-IP assays using FLAG antibody to precipitate *p < 0.05. (D) Mapping of PXR domains/regions for ATF3 binding. 1 µg PXR truncated proteins fused to GST were immobilized onto glutathione agarose beads and incubated with 5 µl in vitro translated ATF3 for GST-pulldown assays. The pulled-down proteins were detected by Western blotting using the ATF3 antibody. (E) Irradiation-induced significant increases in the phosphorylation of threonine/serine in PXR in colon cancer cells. LS180 cells were treated with 10 µM rifampicin (RIF) for 12 h, then radiated at a dose 10 Gy for 4h, PXR antibody was used to immunoprecipitate, then anti- Phosphor - (Ser/Thr) Phe antibody (ab17464) was used to blot. (F) The PXR T432A mutation significantly reduced the binding between ATF3 and PXR as determined by FLAG-IP. H1299 cells were transfected with 1 µg FLAG-PXR wild type or mutants, and 2µg HA-ubiquitin, and/or 1 µg ATF3 as indicated for 2 days, and then subjected to FLAG-IP assays using FLAG antibody to precipitate. (G) The PXR T432A mutation abolished ATM phosphorylation and activation induced by IR in colon cancer cells. LS180 PXR knockout cell was transfected with 1.5 ug PXR wildtype, or mutant for 2 days, and then subjected to 10 Gy of ?- irradiation and harvested after 24 h. The protein levels were determined by Western blotting using the indicated antibodies.