White 384 well plate

The intracellular cAMP level was monitored by the cAMP-Glo assay (Promega) based on the reciprocal relationship between the cAMP concentration and the bioluminescence value. The decreased luminescence reading reflects higher cAMP level in cells. Briefly, 5000 cells (CHO and CHO-GFP-DRD3-FLAG cells) were plated in white 384-well plate (Corning, Manassas, VA, USA, 3570) 24 h prior to the assay. Cells were washed once with PBS and then were pre-treated with 20 μL compounds of interest in PBS for 25 min before treated with 7.5 μL compounds in the presence of 1 mM IBMX, 200 μM Ro 20-1724 and 10 μM forskolin for 15 min at room temperature. The subsequent steps were performed as the manufacture’s protocol indicated. The data were acquired with the Multimode Plate Reader (EnVision, PerkinElmer, Waltham, MA, USA) and analyzed by GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).

2,500 293A-TEAD-LUC cells were plated in 50 μL of growth medium without FBS in white 384-well plates (Corning) and allowed to proliferate for 48 h until fully confluent. 100 nL of compound solvated in DMSO was then transferred to each well using a Bravo Automated Liquid Handling Platform (Agilent) affixed with a pintool head (V&P Scientific). For TEAD-LUC activity assays, 30 μL of BrightGlo reagent solution (Promega, diluted 1:3 in water) was added to each well after 24 h of compound treatment. For cytotoxicity assays, 30 μL of CellTiter-Glo reagent solution (Promega, diluted 1:6 in water) was added to each well after 24 h and 72 h of compound treatment. Luminance values were recorded on an Envision plate reader (Perkin Elmer).

HepG2 cells were plated in white 384 well plates (Corning) at 2000 cells/well in growth medium and incubated overnight at 37°C with 5% CO₂. The next day cells were treated with 100 nL of compound for 24 hr. Cellular ATP levels were measured using CellTiter-Glo (Promega). For testing effects of compounds on mitochondria-dependent oxidative energy production cells were grown in media containing 10 mM galactose instead of 25 mM glucose as main carbon source [30 (link)].

HEK293T-Rex cells incorporating either the ERSE-FLuc or XBP1s-RLuc reporters were plated at 20 μL/well from 250,000 cells/mL in white 384-well plates (Corning). Cell plates were centrifuged for 1 min at 1000 rpm, then incubated at 37°C overnight. The following day cells were treated as described with various concentrations of Thapsigargin or Tunicamycin, incubated for a further 18 hr at 37°C, equilibrated to room temperature, then 20 μL of SteadyLite or Renilla-Glo (Promega, Madison, WI) were added to each well. Luminescence activity was measured 10 min after reagent addition with an EnVision Multilabel Reader (PerkinElmer) using a 100 ms integration time.

A375 cells were stably transfected with the expression vector pGL4.38 [luc2P/p53 RE/Hygro] using TurboFect, as was described previously [37 (link)]. A375-p53-Luc cells were plated in white 384-well plates (Corning) at a density of 2,500 cells/25 μl/well using a Multidrop Combi dispenser (Thermo Scientific) and cultivated overnight. Library compounds were then added using pintool (V&P Scientific) coupled to a JANUS Automated Workstation (PerkinElmer) to a final concentration of 1 μM. The compound library included the Library of Pharmacologically Active Compounds (LOPAC1280, Sigma-Aldrich, Prague, Czech Republic), the Prestwick Chemical Library (Illkirch, France), and the NIH Clinical Trial Collection (NIH, Bethesda, Maryland, USA). A total of 2448 unique compounds were used in the course of HTS screening. Drugs were tested alone and in combination with a DNA damaging drug doxorubicin. The cells were cultivated for 24 h, luciferase production was determined by the One-GLO assay (Promega), and luminescence acquired by an EnVision microplate reader (PerkinElmer).

The kinase activities of rCTR1 and rCTR1-K proteins (20 ng) or Arabidopsis total proteins (3 μg) from 10-d-old seedlings were measured using the STK Substrate 2-biotin in HTRF KinEASE kit according to the manufacturer’s instructions. In brief, assays were conducted in low volume, white 384-well plates (Corning Life Sciences, MA), with a 20 μl assay volume containing 100 μM ATP, 1 μM STK Substrate 2-biotin. For recombinant proteins, liposomes such as 24:0-ceramide, 24:1-ceramide and PA (1 nM, Avanti) were pre-incubated with protein at 4°C for 30 min. For total proteins, Arabidopsis seedlings were non-treated or treated with 10 μM ACC, 50 μM 24:0-ceramide, 24:1-ceramide and PA liposomes and the total proteins were extracted using kinase buffer containing 250 mM (pH 7.0) HEPES, 0.1% NaN₃, 0.05% BSA, 0.5 mM Orthovanadate, 2 mM DTT and 10 mM MgCl₂. The kinase reaction was carried out following incubation at room temperature for 1 h. The reaction was stopped with buffered EDTA followed by the fluorescent development for 1 h at room temperature. The resulting specific TR-FRET signal was detected by a TECAN detection system (Infinite M1000) and calculated based on the fluorescence emission ratio at 665/620 nm.

Test compounds were pre-dispensed into white 384 well plates (Corning). For library screens one compound per well was tested at the indicated final concentration. The following controls were included on the plates: maximum effect control: Amphotericin B (final concentration 2 μM), ATP contamination control: media (± DMSO), cell growth control: DMSO, zero percent effect control: cells at starting concentration. The zero percent effect control wells are left empty during the course of the assay. Media only was dispensed into the ATP-control wells followed by dispensing LdBOB axenic amastigote-like cells at 2 x 10⁴ cells / well to the rest of the wells by using a Wellmate Microplate Dispenser. The assay volume is 25 μl / well. After 72 ± 3 h at 37°C and 5% CO₂ LdBOB axenic amastigote-like cells are added to the zero percent effect control wells at 2 x 10⁴ cells / well followed by read-out with “BacTiter-Glo Microbial Cell Viability Assay” from Promega according to manufacturer’s instructions. Plates were then sealed with clear film and relative luminescence was detected using a plate reader (Victor 3) from Perkin Elmer or PHERAstar FS from BMG LABTECH, with 0.5 s detection time per well). Potency determinations were carried out as library screens with the exception of the following: ten-point curves with one in three dilutions were generated with a top concentration of 50 μM.