Fig. 1: MD-224 reduces PXR protein and transcriptional activity.

A SNU-C4^HiBiT-PXR cells were treated with PROTACs for 2 h and assessed for HiBiT level (n = 1). B SNU-C4^HiBiT-PXR cells were treated with MD-224 for the indicated times and assessed for HiBiT level (n = 6). C SNU-C4^3xFLAG-PXR cells were treated with MD-224 for the indicated times and subjected to western blot using antibodies against FLAG and β-actin. 3xFLAG-PXR was quantified as fold change (FC) relative to the DMSO control at each time point (n = 3). D SNU-C4^3xFLAG-PXR cells were treated with MD-224 for 2 h and subjected to western blot using antibodies against FLAG, MDM2, GSPT1, and β-actin (n = 4). E Chemical structures are shown for MD-224, MDM2 ligand MI-1061, CRBN ligand lenalidomide (LEN), CRBN ligand/linker lenalidomide-propargyl-C₂-NH₂ (LENP), and previously designed PXR PROTAC SJYHJ-040. F SNU-C4^3xFLAG-PXR cells were treated with the indicated compounds for 2 h and subjected to western blot using antibodies against FLAG, MDM2, and β-actin (n = 4). G SNU-C4^HiBiT-PXR cells were treated with MD-224 or SJYHJ-040 for 2, 8, or 24 h and assessed for HiBiT level (n = 4). H SNU-C4^3xFLAG-PXR cells were treated with MD-224 or SJYHJ-040 for 24 h and subjected to western blot using antibodies against FLAG and β-actin (n = 4). I 293T cells were co-transfected with plasmids encoding HiBiT-PXR, LgBiT, and CRBN. After 48 h, cells were mixed with Vivazine substrate and 0.001-10 µM MD-224, and luminescence was measured every 2.5 min for 2 h (n = 3). J SNU-C4^3xFLAG-PXR cells were treated with 5 µM PXR agonist rifampicin (RIF), 10 µM PXR antagonist SPA70, or 0.1-10 µM MD-224 as indicated for 24 h. RNA was extracted and subjected to reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure expression of CYP3A4, PXR, or GAPDH. Data were normalized to 18S RNA and represent FC relative to the DMSO control for each gene (n = 3). For western blots, one representative blot is shown. Error bars represent mean ± standard deviation (SD) from the stated number (n) of biological replicates. Source data are provided as a Source Data file.