L azidohomoalanine aha

Strains were grown overnight in rich medium. The following day, cells were subcultured (1:100) into fresh MM to an OD₆₀₀ of 0.4. Cells were again subcultured (1:4) in MM with and without 1.2 M NaCl. At the indicated time points, cultures were labeled with 400 μM l-azidohomoalanine (AHA) (Click Chemistry Tools) for 30 min. Cultures were treated with 100 μg/mL⁻¹ chloramphenicol at the end of the labeling and collected by centrifugation at 4°C. Cells were washed 3× with ice-cold PBS and stored at −80°C. Cell pellets were thawed and resuspended in a lysis buffer consisting of 1 mg/mL⁻¹ lysozyme, 50 mM Tris-HCl, pH 8.0, 0.5% SDS. Cells were lysed by sonication, and insoluble debris was removed by centrifugation (10 min, 10,000× rpm, 4°C). Covalent attachment of fluorescent tetramethylrhodamine (TAMRA)-alkyne (Thermo Fisher Scientific) to AHA-containing proteins was carried out using a Click-iT protein reaction buffer kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Protein concentrations were determined by Bradford assay. Fluorescent signals in samples were measured in a Synergy H1 reader (BioTek, VT) with 545-nm excitation and 580-nm emission wavelengths. The fluorescence signal was normalized by the protein content of the sample to determine the translation rate. A Student’s t test was performed to determine statistical significance.

Sph-CD were collected at the end of the 72-h detachment period and placed in plates coated with 50 μg/ml poly(2-hydroxyethyl methacrylate) (polyHEMA) to prevent spheroid attachment to the bottom of the plate. Newly synthesized ECM was labeled by incorporating 0.1 mM L-azidohomoalanine (AHA, Click Chemistry Tools), a methionine analog, into the culture medium. AHA was added, such that three separate time groups received AHA for two day pulses over a period of six days. DMEM without methionine was used in these experiments to prevent competition with AHA.
After the two day incubation with AHA, nascent ECM was visualized using fluorescent probe AZDye 488 DBCO (Click Chemistry Tools), which detects azide-tagged biomolecules. Spheroids were stained with 3 μM AZDye 488 DBCO, 5 μM ethidium homodimer-1 (to assess cell viability), 10 μg/ml Hoechst 33342, and 5 μg/ml CellMask DeepRed Plasma Membrane Stain for 30 min at 37 °C. Spheroids were washed twice with PBS and fixed for 15 min in 4% PFA in PBS prior to imaging. As the cells were not permeabilized, any nascent protein detected was extracellular. Images were taken on a Nikon A1RS Confocal Microscope with a 20× 0.75-NA air objective.