SUnSET Assay

Protein translational capacity of ATDC5 cultures (in sextuplicates) was assessed with the SUnSET assay (Goodman and Hornberger, 2013; Henrich, 2016). Puromycin (5.4 μM; Sigma-Aldrich) was incubated for 15 min in the cell culture medium, immediately followed by washing in PBS and fixation for 20 min with 10% formalin (VWR, Radnor, PA, United States). Permeabilization was performed for 10 min with PBS supplemented with 0.1% Triton X-100. Wells were rinsed with PBS with 0.1% Tween (PBS-T) and blocked for 1.5 h with 1% (m/v) skimmed milk powder (ELK, Campina, Zaltbommel, Netherlands) in PBS-T, followed by overnight incubation at 4°C with the primary anti-puromycin antibody 12D10 (Sigma-Aldrich). After washing with PBS-T, wells were incubated for 1 h at room temperature with the secondary goat anti-mouse Alexa488 antibody (Life Technologies). The fluorescent signal intensity was determined using a TriStar² LB942 (Berthold, Bad Wildbad, Germany) equipped with excitation filter F485 and emission filter F535. Fluorescent data were normalized to DNA content from the same well (McCaffrey et al., 1988). To this end, wells were washed with HEPES-Buffered Saline (HBS), followed by 1 h incubation with 5 μg/ml DAPI (Life Technologies) plus 5 μg/ml HOECHST 33342 (Life Technologies) in HBS. After subsequent washing steps with HBS, fluorescent signal intensity was determined using a TriStar² LB942 (Berthold), using the excitation filter F355 and emission filter F460.