DNA damage in Panc-1 cells.

<p>(A) Pan-nuclear γH2AX signals of Panc-1 cells normalized to values of mock treated cells at time points 1 h and 12 h after treatments as surrogates for DNA double strand breaks (n = 3). SFN-treated cells were incubated with either 20 μM or 40 μM SFN for 24 h and afterwards if indicated irradiated with 4 Gy at time point 0 h. (B+C) Western blot of NHEJ pathway proteins after treatment of Panc-1 cells with SFN for 24 h and statistics of their band densities normalized to the loading control. For statistics data of Students t-test are shown (* p<0.05, ** p<0.01, *** p<0.001).</p

Cell cycle distribution in Panc-1 cell.

<p>Cell cycle distribution at different time points: -2 h is the start of 40 μM SFN exposure, 0 h the time point of RT with 4 Gy and the other time points mark 12 h and 24 h after treatment. (A) Example of stacked ModFit curves of Panc-1 cell cycle raw data at the different time points and for the different treatments with G1 and G2 peaks. (B) Relative cell cycle distribution of G1, S and G2/M phase for Panc-1 cells including results of Students t-test of G2/M proportions between control and treatments (* p<0.05, ** p<0.01). (C) Ratios of G2/M and G1 proportion including results of Students t-test in comparison to the respective control (* p<0.05, ** p<0.01).</p

Statistics of G2/M population over time.

<p>Means of relative G2/M population in % (± SEM) with statistics (n = 3; t-test).</p

Sensitizer enhancement ratios (SER).

<p>SERs for an estimated 50%, 10% and 1% surviving fraction for every cell line and each for low and high concentrations of SFN.</p

Clonogenic survival results.

<p>Results of clonogenic survival experiments (n = 3) for each pancreatic cancer cell line after exposure to SFN in a low (2 μM, for MIA PaCa-2 only 1 μM) or high dose (10 μM, for MIA PaCa-2 only 5 μM) for 24 h followed by RT in a low (2 Gy) or high dose (6 Gy), and combination of both. (A) Survival grouped by treatments including Students t-test statistics (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). (B) Logarithmic survival as function of dose for radiation and co-treatments as well as calculated artificial theoretical control curves of assumed isoeffective radiation doses for low (dotted) and high dose SFN (dashed), respectively. Co-treatments with a surviving fraction below the respective theoretical control curve can be considered as supra-additive (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180940#sec006" target="_blank">materials and methods</a> section).</p

Dose-dependent survival fraction of CHO-K1 cells.

<p>Survival fraction (± SEM) of CHO-K1 cells plotted logarithmically with respect to the mean absorbed dose (± SD) determined by three independent clonogenic cell assays including three different doses each (0 Gy, < 2 Gy, > 2 Gy). Sham-irradiated cells are marked with magenta triangles, homogeneously irradiated cells with blue circles and microbeam treated cells by green stars. A linear-quadratic model was fitted to the survival data (blue and green solid lines) to estimate the relative biological effectiveness. The potential saturation of the survival fraction for microbeam irradiation was estimated by the geometry of the tungsten slit array with 6/7 (solid black line).</p

Chromosome aberrations—RBE values.

<p>Relative biological effectiveness (RBE) for equivalent dose effect (number of dicentrics (dic) or centric rings (cr) per cell) of microbeam to homogeneous irradiations based on fitted data.</p

Dose-dependent chromosome aberrations in A_L cells.

<p>Dicentrics (dic) and centric rings (cr) per cell (± SEM) from three individual experiments for mean doses of 0, 1, 1.8, and 2 Gy. Homogeneous irradiation results are shown with blue circles, microbeam irradiation data with green stars and non-irradiated sham data with magenta triangles. Due to its small size, the error bar of the sham irradiation data is not visible at the given scale. A linear-quadratic model was fitted to the data (blue and green, solid and dashed lines). Solid symbols refer to dicentrics, open symbols to centric rings.</p

Schematic drawing of the microbeam setup at the Munich Compact Light Source (MuCLS).

<p>Accelerated electrons (yellow circles) are circulating in the storage ring at twice the rate as two laser pulses (red circles) in the laser cavity. Upon collision of the laser pulse with the electron bunch at the intersection point, a quasi-monochromatic X-ray beam (cyan) is produced. At a source-distance of ∼ 2 m, the tungsten slit array is positioned in the X-ray beam to create a microplanar radiation field. Directly behind the array, cells are situated in a dedicated cell holder. For dose verification, a radiochromic film is placed behind the cell holder. Live dose monitoring is performed with a photon counting detector at ∼ 16 m distance to the source. (Drawing not to scale).</p

Fluorescence microscopy images using the γ-H2AX assay.

<p>DNA double-strand breaks in HeLa cells were stained after (A) microbeam irradiation and (B) homogeneous irradiation with a mean dose of 2 Gy, and (C) no irradiation. Equal acquisition, contrast, and scaling settings were applied.</p