Franziska Hauth, Mahmoud Toulany, Daniel Zips, Apostolos Menegakis

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Cell-line dependent effects of hypoxia prior to irradiation in squamous cell carcinoma lines Franziska Hauth, Mahmoud Toulany, Daniel Zips, Apostolos Menegakis Clinical and Translational Radiation Oncology Volume 5, Pages 12-19 (August 2017) DOI: 10.1016/j.ctro.2017.06.001 Copyright © 2017 Terms and Conditions

Fig. 1 Experimental group O–O–O (normoxia) A) Clonogenic cell survival curves obtained by CFA, B) Residual γH2AX foci dose response, C) mean number of residual γH2AX foci and –lnSF, D) Recalculation of the survival curves based on γH2AX data. Open symbols represent individual experimental values (A-C) or observed CFA points (D). Closed symbols indicate the mean of at least 8 independent experiments for CFA and at least 5 for γH2AX foci assay (A-C) and the calculated surviving fractions (D). Error bars depict the standard error of the mean (A-C) or standard deviation (D). Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions

Fig. 2 Row A) showing experimental group O–H–O (short-term hypoxia during irradiation), Row B) showing experimental group O–O–H (hypoxia after irradiation) and Row C) showing experimental group H–O–O (hypoxia prior to irradiation). For comparison data obtained under normoxic conditions (Fig. 1, O–O–O) are shown as dashed lines and open symbols. First column showing clonogenic cell survival curves obtained by CFA (solid lines and closed symbols), second column exhibits residual γH2AX foci dose response (solid line, closed symbols), and third column showing recalculation of the survival curves based on γH2AX data. The mean of at least 3 independent experiments for CFA and γH2AX foci assay are shown. Error bars depict the standard error of the mean (A-C) or standard deviation (D). Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions

Fig. 3 Linear regression between OER/HMFs estimated from cell survival curves and γH2AX data at 10% survival. Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions

Fig. 4 Bar graph of OER/HMF values calculated for 10% survival based on CFA data. Error bars indicating standard deviation of 3 individual experiments. Dashed line indicating an OER of 1.0, e.g. no disparity between normoxic control and condition tested. Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions

Fig. 5 Representative images of γH2AX foci staining under different experimental conditions. For every cell line (row) and for different experimental conditions (columns) the nuclei are stained with dapi (blue) and the sites of phosphorylated histone H2AX is depicted as green spots. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions

Fig. 6 Western blot analysis of DDR proteins under normoxia (N) or pre-irradiation long term hypoxia (H). Lysates were extracted at the different time points after irradiation. β-Actin and the un-phosphorylated form of the respective proteins served as loading controls. Clinical and Translational Radiation Oncology 2017 5, 12-19DOI: (10.1016/j.ctro.2017.06.001) Copyright © 2017 Terms and Conditions