Phosphate buffered saline (pbs)

For the hanging drop method, a cell suspension (250 or 1000 cells per 50 µL growth medium) was dropped on the inner surface of the lid of a non-coated 90 mm petri dish (nerbe plus GmbH & Co. KG, Winsen, Germany). The lid was turned back on the bottom of the dish filled with 15 mL phosphate-buffered saline (PBS, Bio&SELL) and cultured for up to 7 days (only in 4.2.2. for 9 days). To perform spheroid feeding with culture medium, (after 5 days) lids were turned again and 20 µL medium added per drop. Hanging drop cultures used for qRT-PCR experiments could only be maintained for 7 days since growth medium changes were risky and time consuming in around fifteen plates with 50 drops for each independent experiment.

Bone samples were fixed with 4% formaldehyde (FA, Herbeta) in phosphate-buffered saline (PBS, Bio&Sell GmbH) for 24 h and stored at 4 °C in PBS containing 100 U/ml penicillin and 100 µg/ml streptomycin until further processing. Bone was decalcified with 20% ethylenediaminetetraacetic acid (Carl Roth) solution for multiple days. The bone was then rinsed with tap water, dehydrated with an ascending ethanol series and stored in xylene (Carl Roth) for 24 h, followed by embedding in paraffin/xylene (1:1). 7 µm slices were cut with a microtome (Leica RM2255). Staining with hematoxylin and eosin (both Carl Roth) was performed by the “progressive method” as frequently described [29 (link)].

HUVEC were seeded onto glass coverslips and treated with CPA as described above. Eight days after CPA-treatment, the cells were fixed in 4% paraformaldehyde in PBS (Morphisto GmbH, Offenbach am Main, Germany) for 10 min, permeabilized in 0.25% Triton X-100 (Carl Roth GmbH+Co. KG, Karlsruhe, Germany) for 3 min and blocked in 1 × phosphate-buffered saline (PBS, Bio&Sell GmbH, Feucht, Germany) with 3% bovine serum albumin (BSA, Carl Roth GmbH) for 30 min. Thereafter, cells were first incubated with anti-phospho-histone H2AX mouse monoclonal IgG (Merck Millipore) diluted 1:1000 in 1 × PBS/1% BSA for 1 h at room temperature. Then the cells were incubated with polyclonal Cy3-conjugated goat-anti-mouse antibody (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA) and 0.2 μg/mL DAPI (Carl Roth GmbH) diluted in 1 × PBS/1% BSA for 1 h at room temperature. Immunofluorescence images were obtained with a ZEISS LSM 800 (Carl Zeiss, Jena, Germany). The cell nuclei as well as γH2AX foci were counted using the bioimage analysis software QuPath.