Tristar2

MCMV-Δm157-luc was constructed based on the MCMV-BAC pSM3fr-MCK-2fl [60 (link)] (kindly provided by B. Adler, Munich, Germany). Deletion of the m157 ORF and insertion of the luc sequence into the m157 locus were performed exactly as described [61 (link)]. To quantify organ virus titers, organs were homogenized and virus-tissue suspensions were titrated on a stable mouse embryonic fibroblast cell line. 24 h post titration cells were lysed in luciferase buffer (Roche) and luciferase activity was measured (TriStar²; Berthold Technologies). Virus titer was calculated utilizing a virus standard curve.

The plasmids encoding FRET biosensors were constructed by cloning the coding sequence of mouse normal or Ser68 mutated Panx3 cDNA into the pEYFP-N1 and pECFP-C1 vectors (Clontech). C2C12 cells transfected with biosensors were plated on glass-bottomed dishes and cultured with Phenol-Red-free DMEM containing 10% FBS. Time-lapse FRET imaging was performed on a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging, Inc.). The donor and transfer signals were excited by the 458 nm line of an Argon laser and detected simultaneously between 463–509 nm and 519–620 nm. Images were collected from the middle plane of cells. Time-lapse FRET imaging was captured for 30 s after ATP stimulation. To inhibit ATP receptors, the cells were incubated with 20|U Apyrase before capturing the images. To measure FRET ratio, FRET biosensor vectors including normal or Ser68 mutated Panx3 cDNA transiently transfected C2C12 cells were cultured in a 96-well for 2 days. The FRET ratio was recorded as the 485/536 nm ratio using a plate reader (TriStar^{2 (link)} LB942; Berthold Technologies). 200 µM of ATP stimulation was auto-injected by the LB942.

SW1353 cells were incubated in 6-well plates for 30 min with 25 µCi/mL EasyTag [35S] protein labeling mix (Perkin Elmer, Rotterdam, The Netherlands) in DMEM without methionine or cystine, and supplemented with 10% FCS and 1% P/S. Cells were carefully washed four times with 0.9% NaCl and lysed in 150 µL RIPA buffer. Lysates were sonicated in a water bath and centrifuged for 15 min at 13,200 rpm in an Eppendorf tabletop centrifuge. A quantity of 50 µL of cleared lysates was measured for 5 min on a scintillation counter (Tri-Carb 2910 TR, Perkin Elmer, Rotterdam, The Netherlands), and data were extracted as counts per minute beta. Data were normalized to total DNA content per well using a Sybr Green DNA assay as described earlier [73 (link)]. Alternatively, SW1353 cells were incubated in a 96-well plate for 24 h with 50 µM of the methionine analogue homopropargylglycine (HPG) in DMEM without methionine and cystine, and supplemented with 63 mg/L cystine (Sigma-Aldrich), 10% FCS and 1% P/S. HPG incorporation was detected with a Click-IT reaction (HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit, Thermo Fisher Scientific, Breda, The Netherlands) with a TriStar2 (Berthold Technologies, Vilvoorde, Belgium). Data were normalized to total DNA content per well using the included HCS Nuclear mask blue stain.

For the luciferase assay shown in Supplementary Fig. 1b, the cells were seeded in a 24-well plate at 1.0 × 10⁵ cells/well. After 24 h culture in the absence or presence of 10 nM recombinant Nodal, the cells were washed with PBS and eluted with 150 μl 1 × lysate buffer (Luciferase assay system, Promega). For the intermingled co-culture assay (Fig. 4f; Supplementary Fig. 2), the ligand cells and reporter cells were seeded in a 24-well plate at 1.0 × 10⁵ cells each/well and mixed. After 48 h co-culture, the cells were washed with PBS and eluted with 250 μl 1× lysate buffer. For the measurement of the signal response curve (Supplementary Fig. 7a), the reporter cells were seeded in a 96-well plate at 7000 cells/well. After 1 h incubation, the medium was changed into the fresh medium containing recombinant Nodal and Lefty1. After 48 h culture, the cells were washed with PBS and eluted with 100 μl 1× lysate buffer. The 20 μl lysate prepared above was mixed with 50 μl luciferase substrate (Luciferase assay system, Promega), and the luminescence was measured with a luminometer TriStar² (Berthold technologies).

22Rv1, LNCaP, LNCaP-Pro5, LNCaP-LN3 MR-49F, MR-42D, LAPC4, PC-3, DU145, and WPMY-1 cells were seeded in 24-well plates. Twenty-four hours later, cells were transduced with indicated adenoviruses at a multiplicity of infection of 5. To assess androgen responsiveness, all of the cells were cultured in their respective medium supplemented with 10% charcoal-stripped FBS with or without the addition of 10 nM DHT. Seventy-two hours after treatment, luciferase assays were performed to measure luciferase activity. Briefly, cells were lysed using a passive lysis buffer (Promega, Madison, WI, USA) and luciferase activity was measured by means of either Luminoskan Ascent (ThermoFisher Scientific, Ottawa, ON, Canada) or TriStar² (Berthold Technologies, Oak Ridge, TN, USA) following the addition of D-luciferin, as stated in the Promega luciferase assay system. Relative luciferase activity (RLU) was normalized to total protein amount in each well (RLU = RLU ÷ μg of protein). Protein content was determined by adding 250 μL of Bradford reagent (ThermoFisher Scientific) to 3 μL of total lysate. Absorbance of the corresponding lysate was then read at 595 nm using an Infinite F50 absorbance microplate reader (TECAN, Mannedorf, Switzerland).