Tristar lb942

Cells were seeded in a white 96 well flat bottom plate 1 day prior the treatment. After treatment the medium was removed, the cells washed twice with phosphate-buffered saline (PBS) and the measurement was performed using the luminometric ATP/ADP ratio assay kit (from Sigma-Aldrich MAK135) following the manufacturer’s instruction. The luminescence was measured using microplate reader TriStar² LB 942 (Berthold). The results are expressed as ratio relative levels of ATP on ADP.

The intracellular ATP level was measured using a CellTiter-Glo Luminescent assay kit (Promega, G7570) according to the manufacturer’s instructions. Briefly, Cells were seeded in a 96-well plate 1 day before indicated treatment. After the cells were incubated with the drugs for 2 h, doxycycline was added and cultured for the time indicated in the experiments. Then 100 μL CellTiter-Glo Reagent was added to each well, and then the contents were mixed and incubated at room temperature for 10 min on a shaker. The luminescence was measured using a TriStar² LB 942 multimode microplate reader (Berthold Technologies).

LbNOX, EcSTH, and EcSTH-LbNOX were purified with ANTI-FLAG ® M2 Affinity Gel (A2220, Millipore) in HeLa cells expressing inducible LbNOX-Flag, EcSTH-Flag, and EcSTH-Flag-LbNOX after induction for 24 h (0.1 μg/mL Dox). Enzyme assays were performed in 100 μL assay buffer (50 mM Tris-HCl, pH 7.5, 10 μM flavin adenine dinucleotide, and NADH, NADPH, Thio-NAD + as indicated) at 35 °C for 5 min. Reduction of thio-NAD⁺ was monitored at 405 nm and depletion of NADH (or NADPH) was assessed at 340 nm using a TriStar² LB 942 multimode microplate reader (Berthold Technologies).

To derive BRET² saturation curves, HEK-293 cells were transiently cotransfected with constant amounts of the constructs encoding RLuc8-tagged receptors together with increasing amounts of the constructs encoding GFP²-tagged receptors. For BRET² competition assays, HEK-293 cells were cotransfected with constant amounts of RLuc8- and GFP²-tagged receptor constructs and with increasing amounts of untagged receptors. BRET² assays were performed as described previously [24] (link), [46] (link), [47] (link). Briefly, 180 µl of resuspended cells containing ∼2×10⁵ cells was distributed in 96-well microplates (white Optiplate; Packard BioScience, Meriden, CT, USA). After the addition of coelenterazine 400a to a final concentration of 5 µM using an injector, readings were collected (TriStar LB 942 microplate reader, Berthold Technologies, Bad Wildbad, Germany). Signals at 410 nm (RLuc8 luminescence signal) and 515 nm (emission of light from excited GFP²) were measured sequentially, and 515/410 ratios (BRET² signal) were calculated. The results were expressed in milliBRET units (mBU); BRET ratio × 1000. The expression levels of RLuc8- and GFP²-tagged constructs for each experiment were assessed by total luminescence and fluorescence measurements as described above. Determinations were made in triplicates.

To determine SMAD3 activity, SW1353 cells were transfected with a (CAGA)₁₂ reporter^{33 (link)} and CMV-Gaussia as transfection control^{34 (link)} (100 ng/ul) with Fugene6 (Promega, Madison, WI, USA), according to the manufacturers’ protocol. Bioluminescence was analysed in cell lysates. Cell lysis was performed in Passive Lysis Buffer (Promega) and luciferase activity was measured on a Tristar LB 942 (Berthold) Microplate Reader using the Luciferase Assay System for Firefly luciferase ((CAGA)₁₂) (Promega) and the Gaussia Luciferase Kit (New England Biolabs).

Cell migration assays were performed with a QCM Chemotaxis assay (ECM 510, Millipore) according to protocol recommendations. Briefly, the cells were serum starved for 24 h and 50000 cells were seeded per well. The feeder plate was filled with 0.5% charcoal treated FBS; 10% charcoal treated FBS or 0.5% charcoal treated FBS + E2 (10 nM). Cells were allowed to migrate 8 h or 20 h. After appropriate treatment the cells were detached from the membrane, colored with CyQuant GR Dye. After 15 min of incubation fluorescence was measured (480/520 nm) with a Tristar LB942 (Berthold) with the appropriate filters.

The 47S rDNA gene promotor sequence was custom-made by Genecust, containing nucleotides -1000 up to +60 from the human 47S rDNA transcription start site of the 47S rDNA gene sequence (Ensembl), and cloned into the pNL1.2 vector (Promega, Madison, Wisconsin, USA). The pNL1.2_47S-rDNA promoter plasmid was transfected into SW1353 cells (30.000 cells/cm²; n = 6 samples per condition) with Fugene6. Five hours Post-transfection, cells were incubated for 24 hours with 1 nM BMP7. Post-stimulation, cells were harvested for bioluminescence analysis using cell culture lysis buffer (Promega). Promotor-activity was measured with the Nano-Glo Luciferase Assay System (Promega) on a Tristar LB942 (Berthold Technologies, Bad Wildbad, Germany). Relative differences were determined as compared to control conditions following correction for background and normalization by DNA-content. DNA-content was measured using a SYBR-GREEN assay (Invitrogen).