4.3. Reporter Assay

RBP-J reporter assay was performed using Cignal RBP-J Pathway Reporter Assay (Cat No. 336841; Qiagen, Hilden, Germany) according to manufacturer’s instruction. In the first experiment, TM4 and 15P-1 cells were transfected with RBP-J reporter (a mixture of an inducible RBP-J responsive firefly luciferase reporter and constitutively expressing Renilla construct) and AR siRNA or ZIP9 siRNA. After 16 h cells were treated with T or vehicle. In the second experiment, cells were transfected with RBP-J reporter and after 24 h treated with T, HF or Bic.

Luciferase activity was measured after 24 h using Dual-Glo Luciferase Assay System (Promega) with TECAN Infinite M200 Pro luminometer (Tecan; Männedorf, Switzerland). Firefly luciferase experimental reporter was normalized to Renilla luciferase activity to control transfection efficiency.

Negative controls were performed as follows: (i) RBP-J reporter and non-targeting siRNA; (ii) a mixture of non-inducible firefly luciferase reporter and constitutively expressing Renilla construct (Cignal Negative Control); (iii) Cignal Negative Control and AR siRNA or ZIP9 siRNA; (iv) Cignal Negative Control and non-targeting siRNA. As a positive control a mixture of a constitutively expressing GFP construct, constitutively expressing firefly luciferase construct, and constitutively expressing Renilla luciferase construct was used (not shown).