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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Horm Cancer. 2016 Mar 8;7(3):196–210. doi: 10.1007/s12672-016-0257-2

Fig. 1.

Fig. 1

A-B: LNCaPAR-V7/pLenti cells were transiently transfected with plasmid ARR2PBLuc. After 24 hours in charcoal stripped-FBS (CS-FBS), cells were treated with Doxycycline 0.25 ug/ml. After 24 hours, CUDC was added for 24 hours at concentrations ranging between 0 and 1000 nM. Panel A: percent inhibition of relative luciferase activity in the presence of CUDC-101. B: parallel immunoblot analysis demonstrating the induction of AR-V7 by Doxycicline. Dox (-) represents a control where no dox was added, that was not used in the experiment of panel A. C. Dose response experiment: 22Rv1 were seeded and grown for 24 hrs in CS-FBS, cells were then treated for 24 hours with vehicle or increasing concentrations of CUDC-101 (0 to 0.3 uM) alone, to measure AR-V7 transcriptional activity, or DHT (2 nM) + CUDC-101 (0 to 0.3 uM) to measure flAR transcriptional activity. After 24 hours cells were harvested and aliquots obtained for determination of PSA mRNA. Bars ± SE represent PSA mRNA corrected for 18 S mRNA normalized to cells treated with vehicle. D. Time course experiment: 22Rv1 cells were seeded and grown for 24 hrs in CS-FBS, cells were then treated with vehicle (V) or DHT (D) for additional 24 hours. CUDC-101 (300 nM) was added for 3, 6 or 24 hours in addition to vehicle to measure % inhibition of AR-V7 transcriptional activity (black bars). CUDC-101 (300 nM) + DHT (2nM) were added for 3, 6 or 24 hours to cells previously treated with DHT for 24 hrs to measure % inhibition of flAR transcriptional activity. At the indicated time points cells were harvested and aliquots obtained for determination of PSA mRNA. Bars ±SE represent PSA mRNA corrected for 18 S mRNA normalized to cells treated with vehicle. E: Parallel immunoblot analysis of flAR, AR-V7 and beta actin using aliquots of 22Rv1 cells treated with CUDC101 + DHT from panel C. F: Parallel immunoblot analysis of flAR, AR-V7 and beta actin using aliquots of cells treated with CUDC-101+ DHT from panel D.

**: P<0.01 compared to vehicle treated control. ***: P< 0.001 compared with vehicle + DHT treated control.

Experiments were done in triplicate a minimum of three times.