Streptavidin cy5

Measurement of relative cell growth (CellTiterGlo, 72 h, karonudib (0.0625–1 µM)), viability (propidium iodide, 72 h, karonudib (0.25–1 µM)) and apoptosis (Active Caspase-3, 24 h, karonudib (0.5 µM)) was performed as previously described^{20 (link)}. Proliferation (72 h, karonudib (0.25 µM) was performed using Cell Trace Violet (ThermoFisher Scientific). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed together with cell cycle analysis after 6, 12 and 24 h with karonudib treatment (0.5 µM) as previously described^{20 (link),22 (link),23 (link)}. For cell cycle studies live/dead cell staining (near-IR dead cell stain kit L10119, Thermo Fisher Scientific) was performed prior to fixation. Antibodies: rabbit anti-phospho-histone H3 (pS10 #06-570 1:500; Merck), mouse anti-phospho-γ-histone H2AX (pS139 clone JWB301, #05-635 1:500; Merck), donkey anti-mouse IgG-Alexa488 (#715-545-150 1:500; Jackson Immunoresearch, West Grove, PA), and goat-anti-rabbit IgG-PE (1:500; Thermo). In addition we used biotin-16-dUTP (Merck), streptavidin-Cy5 (PA45001 1:400; GE Healthcare, UK) and Hoechst 33258 (2 µg/ml). Hoechst stained cells were stored at 4°C over night before analysis. Flow cytometry data were analyzed using the online Cytobank flow cytometry software (https://community.cytobank.org)²⁴ or FlowJo v10.

Ten micrograms of biotin-labeled aRNA was fragmented using 5 μl of fragmentation buffer in a final volume of 20 μl and was then mixed with 240 μl of Amersham hybridization solution (GE Healthcare Europe GmbH, Freiburg, Germany) and injected onto CodeLink Uniset Human Whole Genome bioarrays containing 5,5000 human oligonucleotide gene probes (GE Healthcare Europe GmbH, Freiburg, Germany) as described previously (11 (link)). Arrays were hybridized overnight at 37°C at 300 rpm in an incubator. The slides were washed in stringent TNT buffer at 46°C for 1 h, then a streptavidin-cy5 (GE Healthcare) detection step was performed. Each slide was incubated for 30 min in 3.4 ml of streptavidin-cy5 solution, was then washed four times in 240 ml of TNT buffer, rinsed twice in 240 ml of water containing 0.2%Triton X-100, and dried by centrifugation at 600 rpm. The slides were scanned using a Genepix 4000B scanner (Axon, Union City, USA) and Genepix software, with the laser set at 635 mm, the laser power at 100%, and the photomultiplier tube voltage at 60%. The scanned image files were analyzed using CodeLink expression software, version 4.0 (GE Healthcare), which produces both a raw and normalized hybridization signal for each spot on the array. Transcriptomic data have been deposited in Gene Expression Omnibus under the accession number GSE120350.