Figure 6: Effect of tetramerization on the subcellular localization of tRXRα.
(a) Effect of TNFα and K-80003. MCF-7 cells cotransfected with Myc-RXRα, Myc-tRXRα, tRXRα/L433D and p85α were pretreated with or without K-80003 (5 × 10−6M) for 3 h before exposed to TNFα (10 ng ml−1) for 30 min. Cells were immunostained with anti-Myc and anti-p85α antibody, and their subcellular localization revealed by confocal microscopy. (b) Tetrameric tRXRα resides in the nucleus. HEK293T cells cotransfected with Myc-tRXRα were treated with or without K-80003 (5 × 10−6M) for 6 h. Nuclear (N) and cytoplasmic (C) fractions were prepared, subjected to BS3 crosslinking, and analysed by western blotting using anti-Myc antibody. The purity of fractions was examined by analysing the expression of nuclear PARP and cytoplasmic α-tubulin in non-crosslinked fractions. One of three similar experiments is shown. (c) Effect of K-80003 on the growth of MCF-7 cells in mice. Nude mice injected with MCF-7 cells stably transfected with control vector, Myc-RXRα, or Myc-tRXRα were administered with K-80003 (20 mg kg−1) for 12 days. *P<0.05; **P<0.01. (d) Effect of K-80003 on PARP cleavage and AKT activation in MCF-7 xenograft tumours. Lysates prepared from tumours from nude mice treated with vehicle or K-80003 were analysed by western blotting. (e) K-80003 alters the subcellular localization of tRXRα in MCF-7 xenograft tumour cells. Tumour sections prepared from nude mice treated with vehicle or K-80003 were immunostained with anti-Myc antibody. (f) Effect of K-80003 on the growth of MMTV-PyMT mammary tumour. Four-week old MMTV-PyMT mice were fed with or without diet containing K-80003 (100 mg kg−1) for 4 weeks, and the appearance of tumour was determined. **P<0.01. (g) Effect of K-80003 on PARP cleavage and cyclin D1 expression in MMTV-PyMT mammary tumour cells. Lysates prepared from tumours from MMTV-PyMT mice fed with vehicle or K-80003 were analysed by western blotting. (h) K-80003 induces RXRα nuclear localization in MMTV-PyMT tumour cells. Tumour sections from MMTV-PyMT mice fed with or without K-80003 (100 mg kg−1) for 4 weeks were immunostained with ΔN197 anti-RXRα antibody. Scale bar, 10 μm.
