Quantitative DIA proteomics in combination with the transcription inhibitor actinomycin D was performed on the tilapia OmB cell line to identify proteins that are upregulated by transcriptional regulation during hyperosmotic stress. This analysis revealed proteins that are transcriptionally up-regulated by hyperosmolality in these cells. The promoter regions of these proteins were compared and a novel hyperosmolality-induced cis-regulatory element (CRE) and corresponding transcription factor candidate were identified. The CRE was experimentlly validated by site-directed mutagenesis in combination with reporter assays.

Sample preparation by in solution digestion with immobilized trypsin and quantitative proteomics by data-independent acquisition (DIA) liquid chromatography/ tandem mass spectrometry (LCMS2) was performed as previously described [Li, J.; Levitan, B.; Gomez-Jimenez, S.; Kültz, D. Development of a Gill Assay Library for Ecological Proteomics of Threespine Sticklebacks (Gasterosteus Aculeatus). Mol. Cell. Proteomics MCP 2018, 17, 2146–2163, doi:10.1074/mcp.RA118.000973.]. We used the DIA-LCMS2 approach as it enables highly accurate relative quantitation of identical sets of many thousands of proteins in every sample while avoiding undersampling of peaks and inconsistent peak picking.

The tilapia OmB cell line was used for all hyperosmotic stress challenge experiments. OmB cells were maintained in L-15 medium containing 5 % (vol/vol) fetal bovine serum (FBS) and 1 % (vol/vol) penicillin-streptomycin at 26 °C and 2% CO2 as previously described [24,25]. Using a large supply of OmB cell superstock (passage 15; P15), all experiments were conducted on OmB cells between P20 to P27. Cells were passaged every 3-4 days using a 1:5 splitting ratio. Hyperosmotic stress was applied by exposing OmB cells to hyperosmotic medium (650 mOsmol/kg), which was prepared by adding an appropriate volume of hyperosmotic stock solution having an osmolality of 2,820 mOsmol/kg to isosmotic medium having an osmolality of 315 mOsmol/kg. The hyperosmotic stock solution was made by adding an appropriate amount of NaCl to regular isosmotic (315 mOs-mol/kg) L-15 medium. Medium osmolality was checked using a freezing point micro-osmometer (Advanced Instruments). All exposures were performed by acutely increasing medium osmolality from 315 to 650 mOsmol/kg for 24 hours. Parallel handling controls were subjected to medium change without increasing the medium osmolality. Actinomy-cin D, which is a widely-used transcription initiation inhibitor, was applied at a concentration of 10 µM to a subset of hyperosmotically treated OmB cells and isosmotic controls to assess the contribution of transcriptional regulation in the hyperosmotic up-regulation of protein.