AR suppression mediates miR-101 reduction after celastrol treatment.

<p><b>A</b>, miR-101 and AR expressions after celastrol (CEL) treatment. **, <i>P</i><0.01, compared with DMSO. <b>B</b>, Schematic depiction of luciferase reporter constructs as detailed in " <b>Materials and Methods</b>". Wild type and mutant AR binding sites were indicated by italic dash and cross, respectively. <b>C</b>, DU145 cells were transfected with indicated reporter constructs in the presence of pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) plasmid for 24 h, and then luciferase activity was detected. pRLSV40 plasmid was co-transfected for normalization. **, <i>P</i><0.01, between EGFP-AR and EGFP-V transfections. <b>D</b>, ChIP assay. Cell extracts from DU145 or LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or pEGFP-C1 (EGFP-V) were immunoprecipitated with AR antibody or normal mouse IgG. Input was 1/100 of the sonicated chromatin prior to immunoprecipitation. PCR was performed as described in the "<b>Materials and Methods</b>". <b>E</b>, LNCaP cells transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) were treated with celastrol (CEL, 2.0 μM) or DMSO for 3 h, pri-miR-101 and mature miR-101 levels were determined by qPCR. Asterisks denote significance between EGFP-AR and EGFP-V transfections. *, <i>P</i> <0.05; **, <i>P</i> <0.01.