Fig. 4. Loss of TPC2 function alters intracellular drug distribution.

A Lysosomal acidity was assessed by LysoSensor Green staining and flow cytometry. Cells with more acidic lysosomes show increased LysoSensor fluorescence. B VCR-R CEM TPC2 wt and TPC2 ko cells were loaded with LysoSensor Green and fluorescence intensity was analyzed by confocal microscopy (scale bar is 50 µm). C Lysosomal pH was assessed by LysoSensor Green staining and flow cytometry after 30 min of treatment. D Lysosomal volume was assessed by LysoTracker Green staining and flow cytometry after 4 h of treatment. E VCR-R CEM TPC2 wt and TPC2 ko cells were treated with 5 µM doxorubicin in the presence or absence of different concentrations of the lysosomotropic compound ammonium chloride for 24 h. Intracellular doxorubicin fluorescence was analyzed by flow cytometry. F VCR-R CEM TPC2 wt and TPC2 ko cells were treated for 4 h and nuclear doxorubicin (red) was determined by co-staining with Hoechst 33342 (blue) and confocal microscopy (scale bar is 50 µm) and quantified (G) using ImageJ. 119 TPC2 wt and TPC2 ko cells were analyzed in three independent experiments and data points are displayed as box and whiskers with Tukey depiction. H Zoom from (F). Arrows point to the cytoplasmic abundance of doxorubicin (scale bar is 10 µm). Note that brightness and contrast were adjusted to improve visibility (B, F, H). I VCR-R CEM TPC2 wt and TPC2 ko cells were treated with 2.5 µM doxorubicin, and apoptosis was analyzed by propidium iodide staining and flow cytometry. J Immunoblots of VCR-R CEM wt and TPC2 ko cells. K, L Quantitative evaluation of protein levels from (J). Data were acquired from at least three independent experiments. Statistical significance was analyzed by student’s t-test with Welch’s correction (A), two-way ANOVA with Sidak’s posttest (C, D, E, K, L), Mann–Whitney test (G), or two-way ANOVA with Tukey’s posttest (I). Data are shown as mean ± SD, apart from (G).