Fig. 4

Altered properties of acidic vesicles in tpc2⁻ cells. a Intracellular acidic organelles were visualized using a fluorescent LysoSensor™ Yellow/Blue DND-160 probe. Cells were harvested from exponential growth, washed twice with HKC-LoCa buffer and plated onto a 35-mm ibidi μ-Dish at a final cell density at 4 × 10⁵ cells/cm². The cells were allowed to develop for 4 h. The PDMPO probe was added at a final concentration of 2 μM for 15 mins. LysoSensor™ emits predominantly yellow fluorescence, and in less acidic organelles, it emits blue fluorescence [32]. Ratio images are shown and merged with bright field images. Cells were observed using DeltaVision Elite microscope and photographed using an EMCCD camera. Scale bars, 10 μm. b Quantification and statistical analysis (Student t test) of ratios. \( \overline{X\ } \), sample mean; N, sample numbers; ****P ≤ 0.0001. c Ax2 and tpc2⁻ cells were harvested from exponential growth, and 5 × 10⁷ cells were plated on filters incubated on paper presoaked LPS pH 7.0 in the presence of various concentrations of the weak bases imidazole or sodium chloride at 22 °C. Aggregates were observed using a Leica MZ FL III Stereomicroscope and photographed using a Hamamatsu digital camera ORCA-05. Scale bars, 5 mm. One experiment representative of three