Optima plate reader

Protein was extracted 48 hours after doxycycline treatment using M-PER lysis buffer (Thermo) supplemented with 1% phosphatase and protease inhibitors (Thermo). Protein concentrations were determined using BCA assay (Thermo) on an Optima plate reader (BMG Labtech). Proteins were then resolved using 10% Tris-gycine gels (Thermo). After separation, protein (50 µg) was transferred to a PVDF-ImmunoBlot membrane (BioRad) and blocked in 5% non-fat milk for 1 hour at room temperature on a rocking platform. The blots were then incubated with primary antibody overnight at 4°C on a rocking platform. Antibodies were applied as follows: sheep anti-human SIAE (Custom, see antibody production) 3 ug/mL and mouse anti-human β-actin (Sigma). Subsequently, the blots were washed five times in Tris-buffered saline containing 0.05% Tween-20 before and after 1 hour of incubation at room temperature with Licor 800CW infared secondary antibodies. Blots were performed in dupicate. β-actin was used to control for protein loading. Blots were imaged by Licor Odyssey Clx technology using Image Studio 5 software.

Compounds were tested for their ability to release Ca²⁺ in U2OS cells grown in Dulbecco’s modified Eagle medium containing 10% foetal bovine serum at a range of concentrations as indicated (3–4 replicates). Ca²⁺ measurements were performed on confluent cells growing in 96-well plates by incubating with 2 μM fura-2-acetoxymethylester-LeakRes for 45 min in the presence of .1% Pluronic F-127 at room temperature. Fluorescence measurements were performed at λ_ex = 340 nm, λ_ex = 380 nm and λ_em = 526 nm in a Optima plate reader (BMG LABTECH Ltd.). Following data acquisition the 340_ex/526_em fluorescence (in arbitrary units) was divided 380_ex/526_em fluorescence and the data was expressed as a ratio.

Selected bacterial colonies were allowed to grow in 200 μl LB + ampicillin overnight at 37°C in 500 μl 96-well plates (Deepwell plates, Eppendorf) with shaking. The following day, protein expression was induced by adding 0.5 mM IPTG for 2 h. A 50 μl sample of each culture was transferred to new 96-well black plates (Greiner Bio-One), and fluorescence emission was measured in the 485 nm excitation/510 nm emission spectra, using an OPTIMA plate reader (BMG LABTECH). In each plate, 8 wells had cells containing the empty plasmid pT7-3, as a negative control (not fluorescent), and another 8 wells had cells containing the plasmid pEYFP-TP, which expresses the fusion protein YFP-TP (YFP protein fused to the N-terminal end of the TP), and they were used as a positive control (fluorescent). Fluorescence from the selected colonies was measured and its value was normalized by substracting the average of the negative control points and referring it to the positive controls as 100%.

Cells were plated on 96-well plates at a density of 10,000 (SK-N-BE(2)-C, IMR-32, Kelly) or 20,000 (NB8) cells per well and treated as indicated for 72 h. According to manufacturer’s protocol, cells were incubated with reagent for 25 min using the CellTiter-Glo 2.0 kit (Promega, SK-N-BE(2)-C, IMR-32, Kelly) or the CellTiter-Glo 3D kit (Promega, NB8) and bioluminescence was read in an OPTIMA plate reader (BMG Labtech).

HeLa cells, stably transfected with NanoBRET plasmids NanoLuc^®-HDAC6 FL Fusion Vector and NanoLuc^®-HDAC10 FL Fusion Vector (Promega, Madison, WI, USA) were seeded at 20,000 cells/well in white 96-well plates. Without further incubation, tracer (0.3 µM) and drugs were added in separate steps and plates were placed in a tissue culture incubator for 2 h. For NanoBRET quantification, plates were put at room temperature for 10 min. Nanoglow substrate, diluted in OptiMEM without phenol red, was added and measured within 10 min in an OPTIMA plate reader (460 nm emission for donor and 610LP filter for acceptor, BMG Labtech, Ortenberg, Germany). The BRET signal was calculated by the ratio of acceptor signal to donor signal.

If not otherwise declared, cells were precultured for 72 h at a density of 2,000,000 cells per T75 flask (exceptions: Kelly, NB1 and IMR-32 4,000,000 per T75 flask, SJ-GBM2 1,000,000 per T75 flask). Cells were detached with trypsin—EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and seeded in 96-well plates (Greiner, Microplate, 96-Well, PS, F-bottom µCLEAR^®, CELLSTAR^®) in 100 µL medium per well at a density of 5000 (KNS-42, SJ-GBM2, SF188, SK-N-BE(2)-C) or 10,000 (IMR-32, NB1, Kelly) cells/well. HDAC-inhibitor screenings were performed accordingly in 384-well plates (Greiner, Microplate, 384-Well, PS, F-bottom µCLEAR^®, CELLSTAR^®) in 20 µL medium per well at a density of 1250 (SK-N-BE(2)-C, SK-N-AS) or 2500 (IMR-32) cells/well. To avoid edge effects, margin wells (row A and H, column 1 and 12) contained PBS or medium and medium-containing wells were used as background controls. After 24 h incubation, cells were treated for 72 h. Drugs were applied using a Tecan D300e drug printer. Cell viability was quantified using CellTiter-Glo^® 2.0 kit (Promega, Madison, WI, USA). 50 µL CellTiter-Glo^® (96-well) or 25 µL CellTiter-Glo^® (384-well) were added to every well, the plates were shaken at 400 rpm for 5 min and were then incubated for another 10 min. Bioluminescence was quantified on an OPTIMA plate reader (BMG Labtech, Ortenberg, Germany).