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. ß-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.
Fig 2: Immunofluorescence assay detecting MCU, MICU1, MICU2, and Nrf2 expression in CAL 27 cells treated with si-MCU or spermine. (a–c) Representative images of immunofluorescence results. (d) MCU, (e) MICU1, (f) MICU2, and (g) Nrf2 expression was quantified in transfected CAL 27 cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Fig 3: Immunohistochemistry assay detecting MCU, MICU1, MICU2, and Nrf2 expression in CAL 27 cells under different treatments. (a) Representative images of immunohistochemistry results. (b) MCU, (c) MICU1, (d) MICU2, and (e) Nrf2 expression was quantified in transfected CAL 27 cells. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: no statistical significance.
Fig 4: Knockdown of Nrf2 changes MICU1, MICU2, vimentin, and N-cadherin expression induced by MCU activation in OSCC cells. (a) Representative images of western blot results. (b) HO-1 and MCU, (c) MICU1 and MICU2, (d) Nrf2 and vimentin, and (e) N-cadherin and E-cadherin expression was quantified in CAL 27 cells transfected with spermine, sh-Nrf2, or si-MCU. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: no statistical significance.
Fig 5: Immunohistochemistry detecting HO-1, Nrf2, MCU, MICU1, and MICU2 expression in OSCC and normal tissues. (a) Representative images of immunohistochemistry results. Bar value = 20 µm. Magnification: 200×. (b) HO-1, Nrf2, MCU, MICU1, and MICU2 expression was quantified according to immunohistochemistry results. ***p < 0.001; ****p < 0.0001.
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