Overexpression of SidK-fluorescent protein does not affect lysosome degradation or mTORC1 signaling. (A) Dequenching of DQ-BSA (magenta) in control HeLa (top) and RAW264.7 (bottom) cells transfected with GFP (green), where lysosomes had been coloaded with Alexa Fluor 647–conjugated 10 kD dextran (blue). See Materials and methods. Side panels show the individual DQ-BSA and dextran channels of the area denoted by the dotted square, at 1.4× magnification. Images are extended focus compressions of confocal images representative of ≥10 fields from three or more separate experiments of each type. Scale bars: 5 µm. (B) Quantification of DQ-BSA dequenching in SidK^– and SidK⁺ HeLa or RAW264.7 cells, normalized to the 10 kD dextran coloaded into lysosomes. For each condition, three independent experiments were quantified, with ≥10 cells per replicate. Data (magenta circles) are replicate mean ± SEM. P value was calculated using unpaired, two-tailed Student’s t test. n = 14,010, 11,371, 10,955, and 9,864 lysosomes analyzed, respectively. (C) Effects of SidK overexpression on mTORC1 activity. HeLa cells transiently expressing mCherry or SidK-mCherry for 16–24 h were analyzed by immunoblotting for the phosphorylation of mTORC1 targets S6K and ULK1. pS6K-Thr389 (70 kD) is shown in comparison to endogenous S6K (70 kD), and pULK1-Ser757 (140 kD) is shown in comparison to tubulin (55 kD). Blots shown are representative of three independent experiments. Source data are available for this figure: SourceData FS1.