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Figure S1. Western blot analysis of cell-cycle regulatory protein expression in drug-treated DK-MG and SNB19 cells, normalized to β-actin intensity (loading control). Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S2. Western blot analysis of PARP- and cleaved PARP-expression in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios. Figure S3. Western blot analysis of autophagy marker proteins LC3B and p62 in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S4. Western blot analysis of β-actin expression for proteins shown in Fig. 5 and Fig.7 (details in Figs. 5 and 7 legends). Figure S5. Western blot analysis of p-MEK1/2 and p-Erk1/2 in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5). Figure S6. Western blot analysis of Rheb in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S7. Western blot analysis of DNA-repair proteins in drug-treated DK-MG and SNB19 cells. Numbers denote protein/β-actin ratios (details in Fig. 5 legend). Figure S8. Western blot analysis of β-actin expression for proteins depicted in Fig. S7 (details in Fig. 7 legend). Figure S9. Cumulative plots of radius-normalized fc values (fc·a) vs. external conductivity of DK-MG (A) and SNB19 (B) cells were obtained by contra-rotating-field (CRF) technique. Symbols represent mean fc·a (± SD) values from 20 cells measured at ~ 10, 25 and 40 μS/cm. Lines are best fits of Eq. 2 to the data. The steeper line slopes for DK-MG cells (A) imply smaller Cm values compared to SNB19 cells (B). For Cm and CC values, calculated with Eq. 3, see Additional file 2: Table S4. Figure S10. Impact of drug-treatment on cell radius, Cm, and CC values of DK-MG (A) and SNB19 (B) cells. “*” denotes significant difference at p

Supplemental Materials. The primary and secondary antibodies used in this paper. (DOCX 57 kb)

Table S1. Cell cycle-phase distribution in control and irradiated (2 or 8 Gy) SNB19 tumor cells. The cells were fixed either 30 min or 24 h after IR, permeabilized, stained with propidium iodide, and analyzed for their DNA content by flow cytometry. Data are presented as means (± SD) from at least three independent experiments. For detailed description, see legend to Fig. 3. Table S2. Detection of γH2AX as a measure of DNA damage in SNB19 cells by flow cytometry. Mean γH2AX values are normalized to the non-irradiated control (30 min post-irradiation). The data are means ±SE from at least three independent experiments. Table S3. Cloning efficiencies and radiosensitivity parametersa of in vitro irradiated tumor cell lines untreated and pretreated with the MK-2206 and PI-103 either alone or in combination. aMean (± SE) from at least three independent experiments; bCF2 is the colony-forming ability at 2 Gy; cD10 is the radiation dose required to reduce colony-forming ability by 10%; dThe growth inhibition factor IF10 was calculated as (D10 control)/(D10 + inh.). Table S4. Impact of MK-2206, PI-103 either alone or in combination on the area-specific plasma membrane capacitance Cm, the whole-cell capacitance CC* and cell radius. *The data derived from the ROT experiments shown in the Additional file 4: Figure S10 represent the means ± SE of at least 60 cells. (DOCX 63 kb)

Columns represent mean ± SEM of cell proliferation after irradiation shown for the seven cell lines at 21% and 5% oxygen concentration (gray column = with 3-OHB; black column without 3-OHB) (summarized in Fig. 6). The BT20, BT474 and T47D cell lines cultured in the presence of 3-OHB showed a trend towards increased radio-resistance at 21% oxygen (with some significant results at single doses). In contrast, in MCF-7 and MDA-MB 468, 3-OHB cultured cells showed a trend towards impaired cell proliferation following radiation at the same oxygen concentration. At 5% oxygen concentration, 3-OHB seemed to have a sensitizing effect to radiation in some cell lines. Columns represent mean ± SEM of 3 independent experiments with 6 replicate wells per experiment. *

The graphs show the proliferation rate (BrdU; in % of control cells) for the seven different breast cancer cell lines cultured in medium containing 1.5 mM AcAc (white column) compared to control without AcAc (black column) at 5% or 21% oxygen concentration after 5 days of culture (differences are not statistically significant). The columns represent mean ± SEM of data of 4 independent experiments with 3 replicate wells per experiment for each cell line. (PPTX 104 kb)

Graphs present the IC50 with the 95% confidence intervals for the seven tested cell lines obtained for the three cytostatic drugs epirubicin, paclitaxel and carboplatin comparing the IC50 obtained for cells cultured with 3 mM 3-OHB (gray blots) with the control cells grown in medium free of 3-OHB (black boxes). Each blot represents 3–4 independent dose-response experiments with 6 replicate wells per experiment. None of the differences are statistically significant; however a strong tendency to a reduction in IC50 of paclitaxel is seen for T47D grown in 3-OHB medium compared to the control. (PPTX 104 kb)

Characteristics of healthy individuals and RC patients undergoing chemo-radiotherapy (Summary). Table S2. Patients’ characteristics in regard to chemo-radiation toxicities and alcohol/tobacco consumption. Table S3. DNA damage measured by the histone γ-H2AX in PBMCs isolated from blood of apparently healthy donors (N) and unselected rectal carcinoma (RC) patients after exposure to 0.5 or 2 Gy of X-irradiation in vitro or after 5 clinical radiation fractions. Table S4. DNA damage measured by the 53BP1 foci in PBMCs isolated from blood of apparently healthy donors (N) and unselected rectal carcinoma (RC) patients after exposure to 0.5 or 2 Gy of X-irradiation in vitro or after 5 clinical radiation fractions. Figure S1. DNA damage assessed by the mean number of 53BP1 foci in non-irradiated (A) and irradiated (B-D) PBMCs derived from unselected RC patients (triangles), as compared to cells from apparently healthy donors (circles). For further details, see legend to Fig. 1. Filled squares represent the mean values (± SE) for the respective group. “n.s.” indicates that the difference was not highly significant (p > 0.05). Figure S2. Correlational analysis of mean γ-H2AX and 53BP1 foci counts from 500 nuclei per sample. Non-irradiated (A) and irradiated with 0.5 (B and C) and 2 Gy (D) lymphocytes were fixed 30 min (B) or 24 h (C, D) post-IR. The expression of both proteins was analyzed simultaneously at each time and IR points for n = 48 blood samples derived from unselected RC patients. Figure S3. DNA damage assessed by means of the 53BP1 assay in non-irradiated (A) and irradiated (B-D) PBMCs derived from normally-reacting RC patients (grade 0 and 1, up triangles) and radiation-sensitive (grade 2 and 3, down triangles) cancer patients compared to cells from apparently healthy donors (circles). Filled squares represent the mean values (± SE) for the respective group. For details, see legend to Fig. 2. Figure S4. Correlation between the 53BP1 foci expression and tumor staging (see Additional file 1: Table S2). Peripheral lymphocytes were prepared from the blood samples derived from RC patients. Foci counting for 53BP1 were performed in non-irradiated (A), irradiated in vitro with 0.5 and 2 Gy samples 30 min and 24 h post-IR (B and C) or 72 h after 5 clinical radiation fractions (D). Filled squares represent the mean values (± SE) for the respective group. Figure S5. Comparison of the γ-H2AX foci expression in peripheral lymphocytes of RC patients differing in tumor regression grade (TRG, Additional file 1: Table S2). Foci counting for γ-H2AX were performed in non-irradiated (up triangles and circles) cells or after 5 clinical radiation fractions (down triangles and diamonds). Filled squares represent the mean values (± SE) for the respective group. (DOC 1915 kb)

Antibodies. List of primary antibodies used in this study. (DOCX 12 kb)

Colony forming assay data. Colony forming efficiencies and radiosensitivity parameters of tumor cells treated with AZD6244 and NVP-BEZ235 derived with the linear quadratic model. (PDF 128 kb)

Representative cell cycle histograms. Representative DNA histograms of SNB19 and A549 cells treated with AZD6244, NVP-BEZ235, a combination of both drugs and IR with 8 Gy. Cells were fixed 30 min, 24 or 48 h after IR, permeabilized, treated with RNase A, stained with PI and analyzed for DNA content. DNA histograms were deconvoluted with the ModFit Software. (PDF 3250 kb)