Lysosomes can be over-acidified in the presence or absence of TMEM175. (A) Summary of lysosomal pH of WT and TMEM175 KO HEK293T cells with or without apilimod treatment (200 nM, 1 h). Left: Lysosomal pH averaged by cell. The numbers in parenthesis are the number of cells in each condition with each circle representing one cell. Right: Lysosomal pH averaged by independent cell plating and OG loading preparations. Each circle represents one preparation in each respective condition. For HEK293T KO, line TMEM175 KO/5 (Fig. S5) was used. No significant difference in lysosomal pH between WT and KO untreated cells was found, P = 0.78. (B) Summary of lysosomal pH of WT and TMEM175 KO U2OS cells with or without apilimod treatment (100 nM, 2 h). Left: Lysosomal pH averaged by cell. The numbers in parenthesis are the number of cells in each condition; each circle represents one cell. Right: Lysosomal pH averaged by experiment; each circle represents one experiment in each respective condition. No significant difference in lysosomal pH between WT and KO untreated cells was found, P = 0.27. P values are from unpaired two-tailed t test, where * is P < 0.05 and ***** is P < 10⁻⁷. Error bars are SEM.