Colony forming

Dependent on the experiment cells were treated with two different protocols. With the direct seeding protocol exponential growing cells were seeded to 10 cm dishes in adequate amount to be 50–80% confluent next day. Cells were trypsinized, counted and diluted. The dilution was dispensed into different vials and cells were irradiated in suspension. Cells were directly seeded in adequate amounts into 10 cm plates to obtain 100–400 colonies per dish. With the re-seeding protocol exponential growing cells were seeded to 10 cm dishes in adequate amount to be 25–30% confluent next day. The attached cells were treated with different substances or DMSO as a control. 3 h after treatment cells were irradiated with 0, 2, 3, 5, 7, 8 Gy and cultured for 24 h, then cells were trypsinized, counted and re-seeded in adequate amounts into 10 cm plates to obtain 100–400 colonies per dish. For both protocols KP and KPP cells formed colonies after 6 days, BEAS-2B cells formed colonies after 10–11 days. Cells were fixed with ice cold 25% acidic acid in methanol and stained with 0.5% crystal violet. Colonies were count manually. Only colonies containing at least 50 cells were scored. Surviving fractions were calculated by dividing the plating efficiency for the specified dose divided by the plating efficiency of untreated cells. Radiation treatment survival curves were fitted to the linear-quadratic model formula S = exp[-αD-βD²] (S = survival fraction; D = radiation dose; α and β fitted parameters). Curves were fitted and blotted using a non-linear regression and analysed with OriginPro (OriginPro, 2020, OriginLab Corporation, Northampton, MA, USA). Mean survival fractions at 2 Gy (SF2) and 4 Gy (SF4) were also obtained for each cell model and each substance and used to calculate the radiation enhancement ratio at 2 Gy (RER_2Gy) and 4 Gy (RER_4Gy) RER greater than 1 indicates enhancement of radiosensitivity, RER below the value of 1 indicates a radio resistance effect. Similarly, the radiation dose with 25% (D₂₅) and 50% (D₅₀) survival under different conditions was calculated to obtain the dose enhancement ratio (DER₂₅ and DER₅₀) that is calculated by dividing D₂₅ without substance treatment by D₂₅ with substance treatment, respectively D₅₀ without substance treatment by D₅₀ with substance. DER greater than 1 indicates a radio sensitising effect, a DER below the value of 1 indicates a radio protecting effect. Plating efficiency was calculated by dividing the number of colonies by the number of seeded cells. All calculated parameters are listed in Additional file 2: Table S1.