Image studio light version 5.2

ICW analysis was performed as previously described.^{61 (link)} Cells were seeded at 2 × 10⁴ cells/well and treated with increasing doses of CDT (5, 50, 500 ng/ml) or CDT^mut (500 ng/ml) in triplicates in 96-well plates. After 24 h, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min at RT. Upon washing with PBS, paraformaldehyde was neutralized using NH₄Cl (20 mM). Next, cells were permeabilized with 0.2% Triton X-100 in PBS. Following washing with PST (PBS, 0.2% Triton X-100, 2% FCS), cells were blocked with MAXBlock Blocking Medium (Active Motif) supplemented with RNase A (Sigma) and cOmplete Protease Inhibitor (Roche) for 1 h at RT. Cells were then incubated with γH2AX antibody (1 : 200 in PST) for 2 h at RT. As secondary antibody, an infrared fluorescent dye-conjugated antibody (1 : 1000 in PST; 800 nm; LiCor Biosciences, Bad Homburg, Germany) was used for 1 h at RT. For DNA labeling, RedDot2 (1 : 1000 in PST; Biotum, Fremont, CA, USA) was added to the secondary antibody solution. Measurements were performed using an Odyssey Infrared Imaging Scanner (Li-Cor Biosciences). DNA and γ-H2AX staining were simultaneously visualized. Statistical analysis in the Image Studio Light (Version 5.2; Li-Cor Biosciences) was performed to determine the x-fold increase in γ-H2AX signal normalized to the DNA content per well in relation to the control.

Carotid artery smooth muscle cells grown from carotid artery wall explants were grown in 6-well plates. 80–90% confluent cells were washed in PBS and lysed in 1% Lauryl maltoside detergent (Abcam)/PBS plus protease inhibitor cocktail (Sigma), scraped and sonicated. Twenty μg of protein lysates was fractionated on 4–12% gradient polyacrylamide gels and transferred to nitrocellulose membranes (Amersham). Membranes were then blocked in 1:1 Seablock (Thermofisher) and 1:1 Tris-buffered saline plus tween 20 (TBS/T). Membranes were incubated overnight at 4 °C with relevant primary antibody (Supplementary Table 2). Membranes were then washed in TBS/T and incubated with fluorescent secondary antibody (Life technologies; 1:15000) for 2 h. Membranes were then transferred to the LI-COR Odyssey infrared imaging system for visualisation. Densitometric analysis was then performed on LI-COR image studio light (version 5.2).

Cells were lysed in 1 % (w/v) lauryl maltoside detergent (Abcam) in PBS and sonicated. A total of 20 μg of protein lysates was fractionated on 4–12 % gradient polyacrylamide gels and transferred to nitrocellulose membranes (Amersham). Membranes were then blocked in a 1:1 mix of SEA block (Thermo-Fisher) and Tris-buffered saline containing 0.1 % (v/v) tween 20 (TBST). Membranes were incubated overnight at 4 °C with the relevant primary antibody. Membranes were then washed in TBS/T and incubated with fluorescent secondary antibody (1:15000) for 2 hours. Membranes were then transferred to the LI-COR Odyssey-Sa infrared imaging system for visualisation and quantification. Densitometric analysis was then performed on LI-COR image studio light (version 5.2). All primary antibody dilutions are outlined in Additional file 1: Table S2.