FIG. 2.

SphK inhibition enhances 5-epi-induced cell surface exposure of calreticulin in CRC cells. (A) 5-epi dose-dependently induces cell surface exposure of calreticulin (ectoCRT) in DLD-1 cells, after 48 h of treatment, as determined by flow cytometric analysis using PE conjugated anti-CRT antibodies. Inhibition of SphK1/2 using PF-543 (5 μM) enhances 5-epi induced ectoCRT exposure. Results are presented as average MFI normalized to vehicle (DMSO) only controls. (*p<0.0001). (B) 5-epi (7.5 μM) induces ectoCRT in SW-480 and SW-620 CRC cell lines, after 48 h of treatment. PF-543 significantly enhances ectoCRT exposure in SW-480 cells (*p=0.0158). (C) 5-epi (7.5 μM) induces ectoCRT in RKO CRC cells, after 48 h of treatment and PF-543 significantly enhances ectoCRT exposure (*p=0.0007). (D) Representative western blots of CRC cell lines profiling expression of SphK1, SphK2, CRT and GAPDH (individual western blots) using the indicated antibodies. GAPDH was included as a loading control. HEK293 cell lines stably over-expressing SphK1 and SphK2 were employed as positive antibody controls (n=3). CRT, calreticulin; DMSO, dimethyl sulfoxide; ectoCRT, cell surface exposure of CRT; MFI, mean fluorescent intensity; PE, phycoerythrin; SphK1/2, sphingosine kinases 1 and 2.