Envision 2103 multilabel plate reader

14 oligonucleotide gBlocks (IDT), ranging in 500–1000 nt in length, and corresponding to 10 enhancer regions were synthesized. Each gBlock contained a constant 5′ GCTAGCCTCGAGGAT and 3′ ATCAAGATCTGGCCT region, for direct cloning into an EcoRV (NEB) linearized minimal promoter firefly luciferase vector pGL4.23[luc2/minP] (Promega). The resulting reporter constructs were verified by DNA sequencing. BV-2 cells were kindly provided by Dr. Bruce Yankner. N2a cells were purchased from the American Type Culture Collection and maintained following their protocols. Briefly, cells were grown in RPMI-1640 and DMEM respectively, supplemented with 10% fetal bovine serum (FBS) and 1% pen/strep, and split 1:10 every 3 days. Cells were seeded into 24-well plates 1 day before transfection. Transfections into BV-2 and N2a cells were performed with 1 μg of a pGL4.23 plasmid and 200 ng of Renilla luciferase construct pGL4.74[Rluc/TK] (Promega). Luciferase activities were measured 24 h post-transfection using the Dual-Glo Luciferase Assay (Promega) and an EnVision 2103 Multilabel Plate Reader (PerkinElmer) and normalized to Renilla luciferase activity. All assays were performed in triplicate.

For measuring the viability of SRSF1 knockdown cells, cells were subjected to reverse transfection with control or SRSF1-1 siRNA (3 nM) in 6-well plates. After an overnight incubation, cells were seeded in a 96-well plate at 2,500 cells/well, and on the following day, treated with increasing concentrations of VE-821. After three days of ATRi treatment, cells were stained with Alamar Blue (Thermo Fisher) and analyzed with the EnVision 2103 multilabel plate reader from PerkinElmer. For measuring the viability of SETX knockdown cells, cells were seeded in a 96-well plate at 500 cells/well and subjected to forward transfection with control or SETX siRNA (5 nM). After an overnight incubation, cells were treated with escalating doses of ATRi. Four days after the initial transfection, cells were re-transfected with the same siRNA and treated with fresh ATRi to ensure continuous SETX knockdown and ATR inhibition. Four days after the second transfection, cell viability was measured with Alamar Blue as above.

All protein, biotin-dsDNA, and compound stocks were diluted to desired final concentrations in TR-FRET assay buffer (20 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.01% Tween-20, 2 mM DTT) and all experiments were conducted in white, low-volume, flat-bottom, non-binding, 384-well assay plates (Grenier, No: 784904). For CBX8 CD titration experiments, an eleven-point, two-fold dilution series was generated starting at 1.25 μM CBX8 CD. CBX8 CD was incubated with 5 μM compound, 10 nM biotin-dsDNA, 50 nM Lance Ultra ULight anti-6X-histidine antibody, and 2 nM Lance Eu-W8044 steptavidin conjugate at a final reaction volume of 10 μL (0.05% DMSO). Following addition of all assay components, plates were sealed with clear covers, gently mixed on a tabletop shaker for 1 minute, centrifuged at 1000 × g for 2 minutes, and allowed to equilibrate in a dark space for 1 hour before reading. Measurements were taken on an EnVision® 2103 Multilabel Plate Reader (Perkin Elmer) using an excitation filter at 320 nm and emission filters at 615 nm and 665 nm. 615 nm and 650 nm emission signals were measured simultaneously using a dual mirror at D400/D630. TR-FRET output signal was expressed as emission ratios of acceptor/donor (665/615 nm) counts. Data was plotted using GraphPad Prism 9.0.