biostudies-literatureUnknown14(1)Zook EMSH Medical School Hamburg - University of Applied Sciences and Medical UniversityProlonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypotonScientific reports29354https://www.ebi.ac.uk/biostudies/studies/S-EPMC11599563biostudies-literatureDelayed vacuolation in mammalian cells caused by hypotonicity and ion loss.PMC11599563Zook EPan YEKerschbaum HHRitter MClements RJModel MAWipplinger AStauber TfalseDelayed vacuolation in mammalian cells caused by hypotonicity and ion loss.Prolonged exposure of mammalian cells to hypotonic environments stimulates the development of sometimes large and numerous vacuoles of unknown origin. Here, we investigate the nature and formation of these vacuoles, which we term LateVacs. Vacuolation starts after osmotic cell swelling has subsided and continues for many hours thereafter. Most of the vacuoles are positive for the lysosomal marker LAMP-1 but not for the autophagosomal marker LC3. Vacuoles do not appear to have acidic pH, as they exclude LysoTracker and acridine orange; inhibiting the V-ATPase with bafilomycin A1 has no effect on their formation. No LateVacs were formed in cells with a knockout of the essential LRRC8A subunit of the volume-regulated anion channel (VRAC). Since the main feature of cells recovered from hypoton2024-01-01T00:00:00Z2024 Nov2025-04-04T02:38:29.586Z2025-04-04T02:38:29.586ZS-EPMC115995633959271810.1038/s41598-024-79815-z