DSTYK is involved in mitochondrial fitness. (A) Confocal microscopy images. Parental or DSTYK KO H2009 tumor cells were plated, and mitochondria were stained using Mitotracker Deep Red and TMRM and treated with RPMI glucose-free serum to induce stress. Scale bar: 20 µm. (B) Flow cytometry was performed for parental or DSTYK KO H2009 cells in basal and stress conditions and stained with Mitotracker Green and TMRM to assess mitochondrial mass and membrane potential. The results shown are triplicates from a representative experiment out of two performed similarly. Experimental groups were compered by t test. H2009 Mitotracker mean fluorescence intensity (MFI) under stress condition, P = 0.0014; H2009 TMRM/Mitotracker ratio MFI under no glucose condition, P < 0.0001. (C) Confocal microscopy images of lysosomes and mitochondria in parental or DSTYK KO H2009 cells were stained using Mitotracker Deep Red (red) and Lysotracker (green) and treated with RPMI glucose-free serum to induce stress for 4 h. Yellow arrows point to lysosome-enriched areas. Scale bar: 20 µm. (D) Confocal microscopy snapshots zoomed in on mitophagy spots in the time-lapse videos taken of parental or DSTYK KO H2009 cells stained using Mitotracker (green), TMRM (red), and Lysotracker (blue) and treated with glucose-free medium for 4 h to induce stress. Light blue staining is the sum of Lysotracker and Mitotracker. ***, P < 0.001. Representative timeframes corresponding to Video 1. Scale bar: 5 µm.