Dntps

Cells were harvested 24 h after Radon exposure by centrifugation, washed once with PBS and the cell pellets were lysed in 300 µL lysis buffer ML (Macherey-Nagel, Düren, Germany) and stored at −80 °C. RNA was subsequently prepared with the NucleoSpin miRNA Kit (Macherey-Nagel, Düren, Germany) and 500 µg RNA was reverse transcribed using 200 Units M-MLV reverse transcriptase (Promega, Walldorf, Germany), dNTPs (500 µM; Carl Roth, Karlsruhe, Germany) and random hexamer primers (5 µM; Thermo Fisher Scientific, Darmstadt, Germany) for 10 min at 25 °C, 30 min at 37 °C and 30 min at 42 °C.

RNA extraction from cultured cells was performed using the RELIA Prep Kit (Promega Corp., Fitchburg, WI) and the High Pure RNA Isolation Kit (Roche Applied Sciences, Mannheim, Germany), according to the manufacturer’s instructions. For RNA isolation from murine osteoblasts, TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) was used. Complementary DNA (cDNA) was synthesized from 500 ng RNA by reverse transcription using random primers (Thermo Fisher Scientific, Waltham, MA), dNTPs (Carl Roth GmbH, Karlsruhe, Germany), MMLV RT, and RNasin (both from Promega Corp., Fitchburg, WI). Complementary DNA (cDNA) was diluted in nuclease-free water and used for subsequent qPCR analysis. Here, cDNA was mixed with GoTaq Mastermix (Promega Corp., Fitchburg, WI), 10 µM primers (forward and reverse, respectively) and ddH2O. Carboxy-Rhodamine Dye (CXR) (Promega Corp., Fitchburg, WI) served as the reference dye. All qPCR analysis were performed on a StepOnePlus^TM cycler (Applied Biosystems, Carlsbad, CA). Each primer set was validated by melting curve analysis and samples were run in duplicates. Primer sequences are listed in Table S2. Relative mRNA expression of selected targets was calculated using the ∆∆CT method. GAPDH and Actb served as housekeeping genes for human and mouse samples, respectively.

HMVEC cells were cultured under laminar or static conditions in 35 mm dishes, irradiated with 0, 0.1, 0.5, or 1 Gy X-ray or C-ions and stimulated with TNF-α. After 24 h, inserts were removed, washed with PBS, and RNA was isolated using a NucleoSpin miRNA Kit (Macherey-Nagel, Düren, Germany). RNA (500 ng) was reverse transcribed using M-MLV reverse transcriptase (200 U; Promega, Walldorf, Germany), random hexamer primers (5 µM; Thermo Fisher Scientific), and dNTPs (500 µM; Carl Roth) in a ProFlex™ PCR System (Thermo Fisher Scientific) with the settings: 25 °C for 10 min; 37 °C for 30 min; 42 °C for 30 min.

All primers used to generate the templates are shown in Table 1. PCR was performed as follows: 1 U Taq DNA Polymerase and 1X Taq DNA Polymerase Buffer (both from Biozym Scientific GmbH, Hessisch Oldendorf, Germany), 1.0 μM forward and reverse Primer (IDT Integrated DNA Technologies Inc., Leuven, Belgium), 0.8 mM dNTPs (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), 2 ng of template DNA, and RNase-free water (QIAGEN GmbH, Hilden, Germany) to a total volume of 40 μL. PCR was performed in a T3 Thermoblock (Biometra GmbH, Göttingen, Germany) with the following temperature program: initial denaturation step at 94 °C for 300 s, 35 cycles of (40 s at 94 °C, 40 s at 63 °C, 40 s at 72 °C), and a final elongation for 300 s at 72 °C. The purified PCR templates (see “General methods and material” section) were stored at 4 C until further use.