Hx531

Mouse vdr, rxrα, and rxrβ sequences were cloned using the pcDNA6/V5-HisA vector, and mouse genomic DNA sequences for app, bace1, psen1, and the human genomic bace1 sequences were cloned using the firefly luciferase reporter vector pGL4.14 (Promega, USA) or pGL3 with pfu DNA polymerase (Solgent, South Korea). Site-specific mutagenesis and deletion mutants were performed with a QuikChange Site-Directed Mutagenesis Kit (Stratagene, USA) with the gene-specific primers listed in Appendix B Table A2. All sequences were verified. HEK293 cells in 12-well plates were transfected with FuGENE (1–4 μL per 0.5 μg DNA; Roche), and a total of 1.0 μg of DNA (1.0 μg of pGL4.14/pGL3 luciferase reporter, 0.5–1.0 μg of wild-type or mutated pcDNA6/V5-HisA-VDR, pcDNA6/V5-HisA-RXRα, or pcDNA6/V5-HisA-RXRβ, 0.1 μg of pRL-TK-expressing control Renilla luciferase, and the remaining pcDNA6/V5-HisA). HX531 (SML2170, Sigma-Aldrich), a synthetic RXR antagonist, was dissolved in DMSO and added at 2.5, 5.5, and 10.0 μM. Cells were lysed 24–36 h after transfection, and the lysates were rocked at room temperature for 15 min and centrifuged at 8000 rpm for 10 min. A 30 μL volume of supernatant was subjected to luciferase assays with a Centro XS3 LB luminometer (Berhold, Germany). The ratios (firefly/Renilla) of relative luciferase activity were determined.

AM580 (Tocris, #0760, Germany), ER50891 (Tocris, #3823, Germany), CD2314 (Tocris, #3824, Germany), LE135 (Tocris, #2021, Germany), CD437 (Sigma, #C5865, USA), MM11253 (Tocris, #3822, Germany), ATRA (Sigma, #R2625, USA), BMS493 (Sigma, #B6688, USA), GW7647 (Tocris, #1677, Germany), GW6471 (Tocris, #4618, Germany), GW0742 (Tocris, #2229, Germany), GSK3787 (Tocris, #3961, Germany), Troglitazone (Sigma, #T2573, USA), T0070907 (Sigma, #T8703, USA), T0901317 (Sigma, #T2320, USA), SR9238 (Tocris, #5854, Germany), GC1 (Tocris, #4554, Germany), Calcifediol (Tocris, #4036, Germany), SR9011 (Sigma, #SML2067, USA), SR8278 (Tocris, #4463, Germany), CD3254 (Tocris, #3302, Germany), HX531 (Sigma, #SML2170, USA), Testosterone (Aladdin, #T102169, China), Nilutamide (Sigma, #N8534, USA), XCT790 (Sigma, #X4753, USA), Dexamethasone (Sigma, #D4902, USA), Mifepristone (Sigma, #M8046, USA), Corticosterone (Aladdin, #C104537, China), Eplerenone (Sigma, #E6657, USA), Doxorubicin (Solarbio, #D8740, China), Blasticidin (Beyotime, #ST018, China), Puromycin (Beyotime, # ST551, China).