Fig 1.
Inhibition of cell viability by safingol in oral SCC cells.
(A) Oral SCC cells were treated with safingol at various concentrations for 24 h and cell viability was determined using the MTT assay. (B) The effects of safingol on the oral SCC cells lines, Ca9-22 and HSC-3, were also tested at various concentrations and cells were treated for 24 h. Data were means±SD (n = 6). *P<0.05, **P<0.01 compared to untreated control.
Fig 2.
Induction of apoptosis by safingol.
(A) SAS cells were treated with safingol at various concentrations for 24 h, stained with FITC-annexin V and PI, and then subjected to flow cytometry. (B) The percentages of annexin V-negative and PI-negative viable cells, annexin V- positive and PI-negative early apoptotic cells, annexin V-positive and PI-positive late apoptotic cells, and annexin V-negative and PI-positive necrotic cells were determined. (C) Ca9-22 and HSC-3 cells were also treated with safingol, stained with FITC annexin V and PI, and analyzed using flow cytometry. Data were means±SD (n = 3).
Fig 3.
Induction of autophagy by safingol.
(A) SAS cells were treated with 10 μM safingol for 24 h and the expression of LC3-I, LC3-II, Atg5 and beclin-1 were examined by immunoblotting. (B) SAS cells were treated with 10 μM or 25 μM safingol for 24 h. The expression of LC3 was examined by immunofluorescent antibody staining using an anti-LC3 antibody and DAPI. (C) Ca9-22 and HSC-3 cells were treated with 10 μM safingol for 24 h and the expression of LC3 was examined by immunofluorescent antibody staining. (D) SAS cells were treated with 10 μM or 25 μM safingol and stained with acridine orange. Green and red fluorescence was observed under a confocal laser microscope. SAS cells were also treated in combination with 10 μM safingol and 1 mM 3-MA for 24 h. (E) The emission intensity of red (564–627 nm) fluorescence in safingol-treated cells was measured by flow cytometry. (F) Representative transmission electron micrographs of SAS cells treated with 10 μM safingol for 24 h. Many vacuoles are detectable in the cytoplasm of treated cells. Higher magnification of vacuoles reveals mitochondria entrapped inside. Most cells contained intact mitochondria in untreated cells. Scale bar: 2 μm. M: mitochondria, AP: autophagosome, AL: autolysosomes.
Fig 4.
Augmentation of cell death by autophagy inhibitors.
(A) SAS cells were treated with 10 μM safingol and 1 mM 3-MA or 5 nM bafilomycin A1 for 24 h and cell viability was measured by the MTT assay. Data were means±SD (n = 3), *P<0.05. (B) SAS treated with safingol and 3-MA or bafilomycin A1 for 24 h were analyzed using flow cytometry and the proportion of viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells were determined.
Fig 5.
Translocation of endoG by safingol and 3-MA.
(A) SAS cells were treated with 10 μM safingol and 1 mM 3-MA for 24 h and the localization of endoG was examined by immunofluorescent antibody staining. (B) SAS cells were treated with safingol with or without 3-MA for 24 h and proteins in nuclear fraction was examined for the expression of endoG using an immunoblot analysis.
Fig 6.
Involvement of endoG in safingol and 3-MA-induced cell death.
(A) SAS cells were transfected by either endoG-siRNA or nonsense siRNA. Twenty-four hours after transfection, the expression of endoG was examined in the cells by an immunoblot analysis. (B) The expression of endoG in si-RNA-transfected cells was examined by immunofluorescent antibody staining. (C) si-RNA-transfected cells were treated with 10 μM safingol with or without 3-MA for 24 h and cell viability was measured by the MTT assay. Data were means±SD (n = 6), **P<0.01.
Fig 7.
Absence of the effect of a caspase inhibitor on the cytotoxic effect of safingol and 3-MA.
SAS cells were pretreated with the pan-caspase inhibitor, z-VAD-fmk for 2 h, and then treated with safingol and 3-MA in the absence of z-VAD-fmk for 24 h. Cell viability was determined by the MTT assay. There was no significant difference between control and z-VAD-fmk-treated group. Data were means±SD (n = 3).
Fig 8.
Suppression of cell death by the ROS scavenger, NAC.
SAS cells were pretreated with NAC for 2 h and then treated with safingol and 3-MA in the presence of NAC for 24 h. Cell viability was determined by the MTT assay. Data were means±SD (n = 3), **P<0.01.