Fig. 4

LBCS interacts with AR mRNA directly. a Nuclear fraction experiment and real time qPCR detected the abundance of LBCS in the nucleus and cytoplasm. GAPDH is the positive control for cytoplasm, and MALAT1 and U6 is the positive control for nucleus. The results are presented as the means ± SD of values obtained in three independent experiments. b The subcellular distribution of LBCS was visualized by RNA Fluorescent in situ hybridization (FISH) in LNCaP cells. 18S was the positive control for cytoplasm, and U6 was the positive control for the nucleus. Scale bar: 100 μm. c-d Luciferase vectors were constructed containing full length 5′-UTR, different segments of 5′-UTR and 3′-UTR of AR mRNA. The luciferase activity was detected by either co-transfecting control vector or LBCS overexpression vector. The results are presented as the means ± SD of values obtained in three independent experiments. e Potential AR mRNA-LBCS interacting sites were predicted and illustrated. f-i) FRET was performed using a 1:1 mixture of in vitro synthesized LBCS and different AR 5′-UTR RNA regions. j RNA isolation by RNA purification experiment was conducted using LNCaP cells, the different segments of AR 5′-UTR was detected by real time qPCR. GAPDH was detected as a non-specific control. The values are normalized to the negative control LacZ probe and presented as the means ± SD. k Site-directed mutagenesis was conducted on the LBCS interacting site of AR 5′-UTR, the letters in red indicates the mutant base pairs. l The effect of site-directed mutagenesis on the interaction between LBCS and AR mRNA was detected by luciferase assay. The results are presented as the means ± SD of values obtained in three independent experiments. *p < 0.05, **p < 0.01