Glass chamber slides

For immunolabelling of cells, EnSCs (5000 cells) were seeded on glass chamber slides (#94.6170.402, Sarstedt) and cultured in 10% DCC FBS containing DMEM medium. Post treatment with PlGF (20 ng/ml for 6 days) in 2% DCC FBS medium, the cells were fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized for 15 min in 0.1% Triton X-100/PBS. The samples were then blocked with 5% BSA in 0.1% TritonX-100/PBS for 1 h at RT and washed with PBS. The cells were then stained for F-actin with eflour 660-phalloidin (1:1000, #50655905, ThermoFisher Scientific) for 30 min at RT. Post incubation, slides were washed again with PBS, dehydrated, air-dried, and mounted using ProLong Gold antifade reagent containing DAPI (#P36931, Invitrogen). Fluorescence was detected with LSM 800 confocal laser scanning microscope (Zeiss). The images were captured using oil immersion, 40x objective lens. Scale bar - 20 µm. Mean fluorescence intensities were calculated using ImageJ software.

For immunofluorescence microscopy RMS cells and SKMC cells were grown on glass chamber slides (Sarstedt, Nümbrecht, Germany), washed three times with PBS, and fixed with ice-cold Acetone/Methanol (1:1) for 15 min at −20 °C. Subsequently the cells were washed three times with PBS with 0.2 % Tween (PBS-T), incubated for 1h at room temperature (RT) in blocking buffer containing 3% goat serum (in PBS-T; abcam, Cambridge, UK) and exposed for 30 min at RT with anti-TKTL1 mouse monoclonal antibody JFC12T10 (1:150, Zyagnum AG, Pfungstadt, Germany) or anti-DNaseX/Apo10 rat monoclonal antibody (1:50, Zyagnum AG, Pfungstadt, Germany). After three washing steps with PBS-T the cells were incubated for TKTL1 with Alexa Fluor 635 anti-mouse (1:500) and for Apo10 with Alexa Fluor 488 anti-rat (1:750) secondary antibodies (cell signaling, Cambridge, UK) for 1 h at RT. Following three washes with PBS-T all slides were covered with covering medium including DAPI (Invitrogen, Darmstadt, Germany) and images were taken on a Zeiss Apotome (Carl Zeiss Micro Imaging) with an A-Plan 40× ocular.