Figure 8.

HEK293T cells overexpressing TMEM106B-wild-type, but not TMEM106B-D252N, hyper-acidify their lysosomes. (A and B) Fixed J774 macrophages, with their lysosomes labelled with dextran-Alexa405-ApHID, were incubated in various pH-adjusted buffers containing 2.5 µM nigericin and monensin (A), and ApHID/Alexa405 signal ratios were plotted against buffer pH (B). Scale bars = 10 µm. (C and D) HEK293 cells transfected with mApple tagged wild-type or D252N TMEM106B, or empty mApple vector were incubated with dextran-Alexa405-ApHID, which was delivered to lysosomes. mApple is pseudo-coloured blue in the magnified images. The three small panels next to each image correspond to greyscale images indicating Alexa405 (top), ApHID (middle), and mApple (bottom) (D). Red arrowheads indicate enlarged lysosomes with strong ApHID fluorescence. Ratiometric pH imaging was carried out for each condition, and ApHID/Alexa405 ratios were interpolated to pH values using the calibration shown in B. Lysosomal pH values for each condition are shown in C. Scale bars = 10 µm. Error bars in the plots indicate SEM.