Sybr green

The original 3C samples were adjusted with H₂O to approximately 25 ng/μL +/− 10% and, for each adjusted 3C sample, real-time qPCR quantifications were performed to obtain the Ct of each ligation product on 1 μL (containing ~25 ng of DNA). Reaction conditions were as follows (10 μL final reaction volume): 1 μL of sample, 1 μL of primer pair (5 μM each), 1 μL of qPCR mix, and 7 μL of H₂O. The 3C products were quantified in triplicate using a LightCycler 480 II (Roche, Basel, Switzerland) (10 min at 95 °C followed by 45 cycles of 10 s at 95 °C/8 s at 69 °C/14 s at 72 °C) using the Hot-Start Taq Platinum Polymerase (Life Technologies, Carlsbad, CA, USA) and the following qPCR mix [18 (link)]: 0.24% W1 (polyoxyethylene ether W1); 500 µg/mL BSA; 300 µM dNTP; 50 mM KCl; 30 mM MgCl₂; 1/3000 SYBR Green (10,000× in DMSO, LONZA, ref. 50513); 16.24% glycerol; and 400 mM 2-amino-2-methyl-1,3-propanediol buffer mixed to pH 8.3 using HCl. Primer sequences are provided in the Supplementary Table S1.
Quantification values obtained were corrected for potential differences in primer efficiencies and normalised to the “Basal Interaction Level” as previously described [15 (link)], yielding the relative crosslinking frequencies presented in the Figures.

Neutral single-cell agarose-gel electrophoresis was performed for measurement of repair activity of DSBs. Cells were treated with 5 Gy of IR, followed by incubation in culture medium at 37 °C for the indicated times. TREVIGEN comet assay kit (TREVIGEN Instructions) was utilized to detect changed in repair activity. The stained slides with SYBR green (Lonza) were analyzed using a fluorescence microscope (Nikon) at × 400 magnification. The average comet tail moment was scored for 40–50 cells/slide using a computerized image analysis system (Komet 5.5; Andor Technology, Nottingham, UK).

One thousand to 1500 cells were seeded in 96-well plates, cultured overnight, and then incubated in the presence of various concentrations of copanlisib and/or sorafenib. After 6 days, cells were washed with phosphate-buffered saline and underwent osmotic lysis in 100 µl ddH₂O for 45 min at 37 °C. Zero point two percent Sybr green (Lonza, Rockland, ME, USA) was added to each well, fluorescence was measured (GloMax-Multi + Detection System with Instinct Software, Promega, USA) and proliferation index was calculated as a ratio to untreated samples. Three independent experiments were performed per agent, with each data point reflecting triplicate wells. Error bars represented standard deviation of the mean (SD) from three experiments. Data were analyzed by the median-effect method (CompuSyn software; Biosoft, Ferhuson, MO, USA) to determine the drug concentrations resulting in 50% growth inhibition (IC₅₀). The isobologram method and Chou–Talalay CI, a well-established index reflecting the interaction of two drugs, was calculated at different levels of growth inhibition with the use of CompuSyn software^{36 (link)}. CI values of <1, 1, and >1 indicated synergistic, additive, and antagonistic effects, respectively.