DRAM1 knockdown reduces LAMP1 staining of Mm-containing vesicles. (A) Representative examples of LAMP1 (magenta) colocalisation patterns with Mm (cyan), referred to as membrane, punctate, and luminal patterns, at 120 min post infection. Acidification of Mm-containing vesicles was assessed by LysoTracker staining (yellow). Scale bars: 2 μm. (B) Representative examples of LAMP1 (magenta) colocalisation with Mm (cyan), and of the acidification of Mm-containing vesicles, assessed by LysoTracker staining (yellow) in DRAM1 knockdown (KD1-3), as well as control cell lines, at 120 min post infection. The arrowheads indicate colocalisation of LAMP1, Mm and LysoTracker. Scale bars: 2 μm. (C) Percentage of colocalisation of Mm with LAMP1 or LysoTracker in DRAM1 knockdown and control cell lines. (D) Percentage of colocalisation of Mm with LAMP1 in punctate, membrane, and luminal patterns, in DRAM1 knockdown and control cell lines. (E) Percentage of colocalisation of LysoTracker with LAMP1-negative Mm and LAMP1-positive Mm punctate, membrane, and luminal Mm patterns, in DRAM1 knockdown and control cell lines. Bar graphs show the data from three independent experiments, where the mean of each replicate is indicated with a colored symbol. In total, 18 ROIs were analysed per control or DRAM1 knockdown (KD1-3) group, with the following total numbers of Mm observations per group: Ctrl = 479, KD1 = 473, KD2 = 469, and KD3 = 502. Significance was assessed by beta-binomial logistic regression with Dunnett’s multiple test correction. Error bars represent the standard deviation of the three independent replications of the experiment (* p < 0.05; ** p < 0.01; *** p < 0.001).