Figure 3. Negative interaction between PRSS8 and Sphk1/S1P/Stat3/Akt signaling in colorectal cancer cells, in Sphk1 mouse models and in human colorectal cancers.
A. knockdown of PRSS8 led to the upregulation of Sphk1 in HCT8 cells. B. overexpression of Sphk1 in HCT8 cells suppressed PRSS8 expression. C. knockdown of Sphk1 expression caused upregulation of PRSS8 in HCT116 and SW480 cells. D. PRSS8 expression was increased in Sphk1-/− mouse colon (lower panel), compared to the Sphk1+/+ mice (upper panel). E. Online gene profile data set (www.oncomine.com) was deeply mined and neutralized, the results showed that PRSS8 was higher and Sphk1 was lower in normal colorectal mucosa, but RPSS8 was reduced (p=1.8×10−5) and Sphk1 was increased in colorectal cancers (p=9.1×10−28, Figure 3E), exhibiting a strong negative correlation. F. the interaction between PRSS8 and Sphk1 was examined by co-immunoprecipitation assay. PRSS8/Sphk1 complex was precipitated using anti-Flag or anti-Sphk1 antibodies, probed with anti-Sphk1 or anti-Flag antibodies, respectively. G. The interaction between PRSS8 and Sphk1 was also confirmed by immuno-fluorescence staining assay, showing that PRSS8 protein (p-EGFP-PRSS8, Green-fluorescence staining) was co-localized with Sphk1 protein (pEx-6-Sphk1, Red-fluorescence staining) in cytoplasm and nuclei.