Thereafter, cells were treated for 48 h with the indicated inhibitors or vehicle (DMSO) before the MTT reagent was added for 5 h and the absorbance of formazan was measured using a SpectraMax M2 microplate reader (Molecular Devices, Bucher Biotech AG, Basel, Switzerland)

Thereafter, cells were treated for 48 h with the indicated inhibitors or vehicle (DMSO) before the MTT reagent was added for 5 h and the absorbance of formazan was measured using a SpectraMax M2 microplate reader (Molecular Devices, Bucher Biotech AG, Basel, Switzerland). Colony Formation Assay Cells were cultured in 60 mm diameter dishes at a density of 700 cells per Tlr4 dish in cell culture medium. (2.7M) GUID:?4ED6EEED-5E8E-416F-9A54-DA882A5F8125 Figure S2: PKC isoenzyme expression in MDA-MB-231 cells. Lysates were prepared from MDA-MB-231 wt, SK-1 kd and SK-1 ov cells, and analyzed for PKC isoenzymes by Western blotting using specific antibodies Disulfiram against the various PKC proteins (dilution 11000) or GAPDH (dilution 12000) as loading control.(EPS) pone.0039209.s002.eps (4.6M) GUID:?CB152E48-274A-4750-8313-76211DFD5185 Figure S3: Inhibition of SK-1 and sphingosine treatment increases cells in SubG1 and with 4N and 8N DNA content. (A) MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 3105 cells per dish in cell culture medium. After 24 h, cells were treated with combination of SK I II inhibitor and sphingosine, DMSO was used as vehicle control. After another 24 h, cells were fixed, stained with propidium iodide and the DNA content was measured by circulation cytometry using a FACSCalibur circulation cytometer and the Cell Mission software for data processing. Data are means S.D. (n?=?3); Disulfiram *p 0.05, ***p 0.001 compared to the DMSO control values. (B) MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 3105 cells per dish in cell culture medium. After 24 h, cells were treated with the SK I II inhibitor or sphingosine, DMSO was used as vehicle control. After another 24 h and 72 h, cells were fixed, stained with propidium iodide and the DNA content was measured by circulation cytometry using a FACSCalibur circulation cytometer and the Cell Mission software for data processing. Data are means S.D. (n?=?3); *p 0.05, **p 0.01, ***p 0.001 compared to the DMSO control values.(EPS) pone.0039209.s003.eps (1.3M) GUID:?64FE0B49-2B02-4F19-A03A-C8FCCB29A710 Figure S4: Downregulation of SK-1 in MDA-MB-231 cells does not affect expression of Beclin 1 and Aurora B. Lysates were prepared from MDA-MB-231 wt and SK-1 kd cells, and analyzed for Beclin 1 (Atg 6) (dilution 11000), Aurora B (dilution 11000) and GAPDH (dilution 12000) as loading control by Western blotting using specific antibodies.(EPS) pone.0039209.s004.eps (4.0M) GUID:?2DCC1ECA-BE6B-4795-9298-6837F66DC47F Abstract Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and comparable effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells joined mitosis but failed to divide, and in Disulfiram the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for malignancy therapy. Introduction The cellular sphingolipid signaling pathway is usually a highly conserved balanced system comprising ceramide and sphingosine with proapoptotic functions on the one hand, and sphingosine-1-phosphate (S1P) promoting cell survival and proliferation on the other hand [1]. Elevated S1P favors.