Alkalinization of TMEM175 KO cells with LysoTracker. Measured relative fluorescence from cells loaded with LysoTracker, a non-ratiometric, pH-sensitive dye which increases in fluorescence with increased [H⁺] or decreased pH. Data were collected in WT and TMEM175 KO pairs for both the fed and starved states. The analyzed LysoTracker intensities were normalized to the value averaged from WT in the same pair. (A) LysoTracker data from macrophages. Left: Representative fluorescence signal of WT cells and TMEM175 KO cells in a single field of view with a 20 μm scale bar. Right: Summary of relative LysoTracker fluorescence analyzed by cell in fed and starved WT and TMEM175 KO cells. Numbers in parenthesis are the number of cells analyzed per condition and the circles represent the normalized intensity of one cell. (B) Summary of relative LysoTracker intensity analyzed by individual lysosomes and averaged per cell in fed and starved WT and TMEM175 KO hippocampal neurons. Sequence deletions in three independent TMEM175 KO mouse lines (K1, K3, and K6) were used and respectively labeled. Each circle is one cell and over 300 lysosomes were counted in each condition and the numbers in parenthesis are the number of cells. The only significant difference observed is the decrease in fluorescence in TMEM175 KO cells after starvation in both hippocampal neurons and macrophages, consistent with lysosomal alkalinization in the KO. P values are from unpaired two-tailed t test, where * is P < 0.05 and error bars are SEM.