Figure 3.

. SCaMC-1 selectively protects from oxidative stress-induced cell death. (a) Left panel: Control and SCaMC-1-KD 143B cells were incubated with H₂O₂ for 6 h and cell death was evaluated by confocal microscopy after 1 μM calcein-AM and 2 μM PI staining. Right panel: Control and SCaMC-1-KD COS-7 cells were incubated with menadione for 2 h and cell death was evaluated by flow cytometry after PI staining (mean±S.E.M., n=6 and 5, respectively; ^*P<0.05). (b) Control and SCaMC-1-KD 143B cells were incubated with 1 mM H₂O₂ for 3.5 h or with 100 μM menadione for 1 h and cell death was evaluated by flow cytometry after TMRM and Annexin-V–FITC staining. A representative experiment is shown where H indicates the percentage of healthy cells (positive for TMRM and negative for Annexin-V, delimited by the quadrant) and D indicates the percentage of dead cells (negative for TMRM and positive for Annexin-V). The number at the bottom of each panel indicates the percentage (mean±S.E.M., n=6) of healthy cells. ^**P=0.00031; ^*P=0.014. (c) Control and SCaMC-1-KD COS-7 and 143B cells are equally sensitive to 1 μM staurosporine-induced cell death. Cells were treated with the drug for 0, 16 or 40 h, and death was evaluated by flow cytometry after PI and Annexin-V–FITC staining. (d) SCaMC-1-KD cells are more vulnerable to phototoxicity-induced oxidative stress. Control and SCaMC-1-KD COS-7 cells were loaded with 100 nM TMRM; illuminated with 10% laser power of the 543 nm He–Ne line of the confocal system; and collapse of mitochondrial ΔΨ was monitored over time. Lower left panels: SCaMC-1-KD cells were treated with 100 μM EGTA and 1 μM thapsigargin. Lower right panels: Cells treated with 5 μM CsA and 50 μM BKA. The figure shows representative images of at least three independent experiments. (e) Sensitivity of SCaMC-1-overexpressing 143B SCaMC-1-KD cells (left panel) and liver clone-9 cells (right panel) to mPT-dependent cell death. Cells were transiently transfected with GFP, or with GFP and SCaMC-1, and exposed to C₂ ceramide or H₂O₂ for 6 h. The bar shows the change in the percentage of GFP fluorescent cells (as compared with total cell number) after the treatment.²¹ An increase in this percentage as compared with control transfections indicates protection by SCaMC-1. The right panel shows the increase in SCaMC-1 expression after the transfection. SCaMC-1^*: Rescue construct with synonymous mutations (see the Supplementary Materials and Methods). (f) Control and stable SCaMC-1-overexpressing liver clone-9 cells were incubated with H₂O₂ for 3 h and cell death was evaluated by flow cytometry after PI staining (mean±S.E.M., n=3; ^*P<0.05). The right panel shows the increase in SCaMC-1 expression in the stable SCaMC-1-overexpressing clone