Additional file 1 of Opposite effects of the triple target (DNA-PK/PI3K/mTOR) inhibitor PI-103 on the radiation sensitivity of glioblastoma cell lines proficient and deficient in DNA-PKcs
Creators: name: Djuzenova, Cholpon S.
name: Fischer, Thomas
name: Katzer, Astrid
name: Sisario, Dmitri
name: Korsa, Tessa
name: Steussloff, Gudrun
name: Sukhorukov, Vladimir L.
name: Flentje, Michael
Publisher: name: figshare
Year: 2021
Subjects: subject: Biophysics
subject: Biochemistry
subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: 69999 Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology

description: Additional file 1: Supplemental Materials. The primary and secondary antibodies used in this paper. Supplemental Methods.: Comet assay and staining for HDAC5 and DAPI. In order to characterize the tested cell lines, preliminary experiments were carried out via Comet assay. The Comet assay was performed under alkaline conditions following a protocol reported elsewhere [19]. Just before irradiation, cells were embedded in a thin layer of agarose spread on glass microscope slides. The slides were placed on ice, subjected to irradiation and transferred immediately either into ice-cold lysis buffer or in CGM for the indicated times. DNA fragmentation was quantified from the “Tail Moment” (TM, given in arbitrary units, a.u.) defined as the product of the percentage of DNA in the comet tail and the tail length. Supplementary Fig. S1 shows the induction and repair of DNA damage measured by the Comet assay in the cells of both cell lines immediately and up to 40 min after irradiation with 5 Gy. As seen in Supplementary Fig. S1, the initial TM was higher in MO59J cells, however, the residual DNA damage was similar in both cell lines, as were the kinetics of DNA damage disappearance. Supplementary Fig. S2 shows the distribution of TM in both cell lines measured immediately after IR. As seen in Supplementary Fig. S2, the histogram of TM values in intrinsically radiosensitive MO59J line was shifted towards higher DNA damage. It is worth mentioning that DNA damage measured by the alkaline Comet assay includes single-strand breaks and alkali-labile sites and base exchanges. Another preliminary test to characterize intrinsically radioresistant MO59K and radiosensitive MO59J cell lines was staining for HDAC5 protein and subsequent HDAC5 foci counting (Supplementary Fig. S3). To this end, cells were cultured on microscope glass slides for at least 24 h and stained with anti-HDAC5 (1:200) primary antibodies essentially as described elsewhere [57] with the exception of primary antibodies. For each experiment or cell line, at least 100 nuclei were examined and HDAC5 foci were scored by eye at a magnification of 1000x. Probes were then quantified by counting the number of foci per nucleus. As seen in Supplementary Fig. S3, HDAC5 foci were much more expressed in radioresistant MO59K cell line. Simultaneously slides were counterstained with DAPI (Supplementary Fig. S4). Fig. S1. DNA damage (Tail Moment, TM) induction and repair measured by the comet assay in human GBM cell lines irradiated with 5 Gy of X-rays in vitro. Immediately after irradiation, the samples were placed at 37 °C in a 5% CO2 incubator. At the indicated time intervals after X-ray exposure, the cells were lysed and subjected to the alkaline comet assay. Up to 75 cells were analyzed for each slide. Each point (bar) represents the mean value (± SE) of TM for the respective time point. The curves are best least-square fits of exponential decay function to the datapoints. Fig. S2. DNA damage (Tail Moment) induction measured by the comet assay in human GBM cell lines MO59K and MO59J irradiated with 5 Gy of X-rays in vitro. The cells taken at the indicated time (2 min) after X-ray exposure were lysed and subjected to the alkaline Comet assay. Up to 75 cells were analyzed for each slide. The curves are best least-square fits of normal distribution to the datapoints. Fig. S3. Representative histograms depicting the HDAC5 focus formation in control non-irradiated MO59K and MO59J cells. Cells were analyzed for HDAC5 focus formation 24 h after seeding. About 100 nuclei were counted per each cell line and experiment (n = 3). Fig. S4. Representative histograms depicting the mean DAPI fluorescence intensity per nucleus in control non-irradiated MO59K and MO59J cells. Cells were cultured on the slides and 24 h after seeding stained with DAPI. Fluorescence intensity was quantitated with the ImageJ program in about 100 nuclei counted per each cell line and experiment (n = 3). Fig. S5. Cellular viability measured by an ATP test. Changes of intracellular ATP in 2 tumor cell lines exposed to serial dilutions of PI-103 for 24 h. ATP content was measured by standard luciferase bioluminescence assay. Quadruplicate data derived from at least three independent experiments were averaged, normalized against non-treated controls (DMSO) and analyzed using the standard four-parameter logistic model to generate dose-response curves. Error bars indicate SD values. Fig. S6. Effects of radiation, PI-103 or combined PI-103-radiation treatments on colony-forming ability of MO59K cells. One thousand MO59K cells were plated on Petri dishes for 16 h, treated with radiation (A: 2, 3, and 5 Gy), or with PI-103 (B: 0.5 μM, 1 μM, and 2 μM) or a combination of radiation (C: 3 Gy) and PI-103 (0.5 μM, 1 μM and 2 μM)) added 3 h before irradiation. Twenty four hours post-irradiation the inhibitor was washed out and the cultures were incubated in fresh CGM for the next 12 days, fixed and stained with crystal violet. The numbers of colonies were counted and averaged from three independent experiments. Combination index was determined according Malyarenko et al. (2020) and Chou (2010) and found to be at tested concentrations of PI-103 and radiation dose of 3 Gy