Lack of TMEM175 results in lysosomal alkalinization, not hyper-acidification, in mouse hippocampal neurons. (A) Sequence deletions in two independent TMEM175 KO mouse lines (K1, K3) used to determine lysosomal pH. Left: Sanger sequencing chromatograms with the deletion site in each allele indicated by an arrow. Right: sequences deleted in exons 3 (green), exon 4 (blue), and the intron between (lower case). Numbers of base pairs deleted in exons 3 and 4 in each allele are K1 (20 bp in exon 3 and 47 bp in exon 4) and K3 (24 bp in exon 3 and 49 bp in exon 4). Another line (K6, used in Fig. 4 and Fig. S3 B) has 23 bp deletion in exon 3 and 51 bp in exon 4. Partial sequences of WT in the region are 5′-TTC​TAA​TCG​TAA​CTG​TGG​CCT​GGA​CAG​CAC​ATA​CCA​GGT​AG-3′ 2.3 kb 5′-TTA​GAT​TGT​TCC​AGG​TTG​TTG​GAA​AAA​TAG​ATG​ATA​CAC​TTG​CCT​TGC​TCA​ACC​TGG-3′ (exon sequences are in capital; sequences targeted by the two guide RNAs used for the generation of KO mice are underlined). (B) Average ratios of fluorescence signal, before calibration, from excitation at 490 nm to that at 440 nm in OG-loaded hippocampal neurons cultured from WT and KO mice, with and without starvation. The starvation condition for neurons is overnight incubation without B-27 supplement. The number in parentheses is the number of cells with each circle representing the ratio for one cell and the corresponding mouse line (K1 or K3) is labeled. (C and D) Summary of the lysosomal pH of WT and KO hippocampal neurons cultured from two KO lines with (D) and without (C) starvation. The numbers in parentheses are the number of cells for each condition. Each circle represents the average of one neuron. See Fig. S4 A for lysosomal pH averaged from each individual animal. P values are from unpaired two-tailed t test, where **** is P < 10⁻⁶ and ***** is P < 10⁻⁷. Error bars are SEM.