Fig 1: Inhibition of cathepsin S redistributes Ca2+ levels and alters MCU complex expression in glioblastoma cell lines. Cells were treated with 20 μM ZFL for 48 h. In the ZFL+siMCU groups, siRNA targeting MCU was added. (A, B) Cells were stained with Fluo-2 or Rhod-2 as previous described by flow cytometry. Then intracellular Ca2+ (A) and mitochondrial Ca2+ (B) were measured by flow cytometry. (C, D, F, G) The expression of MCU, MICU1, MICU2 and EMRE among the control groups, shCtrl groups, and shCTSS groups and groups treated with different concentrations of ZFL was measured by western blotting. β-Actin was used as an internal control. (E, H) Protein levels of MCU after adding siMCU were accessed by western blotting. (I, J) Then, intracellular and mitochondrial Ca2+ was imaged by the fluorescence staining of Fluo-2 and Rhod2. Scale bar, 50 μm. Data are represented as the means ± SEMs of three independent experiments. *p < 0.05, **p < 0.01 versus the control group.