1 sirna

LNCaP cells in steroid hormone-depleted medium without phenol-red, containing 10% CS-FBS were prepared for transfection. One × 10⁵ LNCaP cells were re-suspended in 100 μl resuspension buffer R (Neon® Transfection System; Invitrogen, Carlsbad, CA, USA) with 2 μM siRNA for HSD3B1 (s6926; Silencer® select, Ambion) or control non-silencing siRNA (#1 siRNA; Ambion) and transfected in 100 μl Neon tip with Neon transfection system (Invitrogen) using two pulses (1250 V input pulse voltage/20 ms input pulse width). Five × 10⁴ transfected cells in 500 μl phenol-red free medium containing 10% CS-FBS with 10 nM 3β-diol were plated each well of 24 well plate in triplicate and cultured for 3 days before Tandem-R PSA tests.

DLG2 (NM_001364) and DLG2 (NM_001351274.2) overexpression plasmids on a backbone of pcDNA3.1/C-(K)-DYK (OHu25658D and OHuq102626D respectively) vector were purchased from GenScript. LIN7A (NM_004664) over expression plasmid on a backbone of pCMV6-AC-GFP (PS100010) was purchased from Origene (RG221902). siRNA targeting DLG2 (s4122), LIN7A (s16836) or Silencer™ Select Negative control No. 1 siRNA (4390843) were purchased from Ambion (Thermo Fischer Scientific). SKNAS and HEK293 cells were grown to 80% confluence and subsequently transfected with; DYK-tagged DLG2 isoform 7, DYK-tagged DLG2 isoform 2, combined with GFP-tagged LIN7A, empty vector “mock” (pCMV6-Ac-GFP), si-LIN7A or scrambled negative control “mock”. 100 ng of DNA or 10 pmol siRNA was complexed with 0.3 µl of Lipofectamine 2000 according to the Lipofectamine 2000 reagent forward transfection protocol (Invitrogen; Thermo Fisher Scientific, Inc.).