Triton x 100 pbs

4 x 10⁵ cells per well were grown in 3D lrECM for 23 h, then treated with different levels of BEMER therapy (~13 μT and ~35 μT; 8 min) and irradiated 1 h later with 6 Gy or left unirradiated. After 24 h, cells were isolated using PBS and trypsin (PAA), fixed with 3% formaldehyde/PBS (Merck, Darmstadt, Germany), permeabilized with 0.25% Triton-X-100/PBS (Roth, Karlsruhe, Germany) and stained with specific antibodies for γH2AX and 53BP1. Samples were spread on a slide and covered with Vectashield/DAPI mounting medium. γH2AX/53BP1-positive foci were counted microscopically with an Axioscope 2 plus fluorescence microscope (Zeiss) and defined as residual DSB [34 (link)]. Immunofluorescence images were sustained using LSM 510 meta (Zeiss).

Synemin-depleted un- and irradiated SAS cells cultured for 24, 48 and 72 h were incubated with 10 mM BrdU (BD Biosciences) for 10 min. Cells were harvested using 1× trypsin/EDTA, fixed in ice cold 80% ethanol for 10 min and incubated for 10 min with 0.01% RNase A (Sigma-Aldrich), followed by 30 min incubation with 2 N HCl (Sigma-Aldrich) and 0.5% Triton-X-100/PBS (Roth, Karlsruhe, Germany). Subsequently, mouse anti-BrdU antibodies and propidium iodide (Sigma-Aldrich) were added for the detection of incorporated BrdU and total DNA content. Cell cycle distribution was determined using an FACS Calibur (BD Biosciences) and analyzed using the FlowJo software (version 7.6.2).

Keap1 depleted UTSCC14 cells were irradiated with 6 Gy X-rays and cultured for 6 and 24 h later on (non-specific RNA as control). Then, cells were incubated with 10 mM BrdU (BD Biosciences) for 10 min and harvested using trypsin/EDTA solution. Thereafter, cell suspension was fixed with ice-cold 80% ethanol for 10 min and incubated for 10 min with 0.01% RNase A (Sigma-Aldrich) followed by 30 min treatment with 2 N HCl (Sigma-Aldrich) and 0.5% Triton-X-100/PBS (Roth). Subsequently, mouse anti-BrdU antibodies and propidium iodide (Sigma-Aldrich) was added for the analysis of BrdU incorporation and total DNA content. As previously published⁴⁶ , Cell cycle distribution was determined using a FACS Celesta (BD Biosciences) with FlowJo software (version 7.6.2).