Fig 1.
Overexpression of PRL-3 widely increased protein phosphorylation.
(A) Confirmation of PRL-3 overexpression in HCT116 and LoVo cells. (B) Several phosphoproteins increased by PRL-3 in HCT116 and LoVo cells. (C) PRL-3’s wide impact on the whole protein phosphorylation, including tyrosine phosphorylation and serine/threonine phosphorylation in HCT116 and LoVo cells. (D) PRL-3’s impacts on phosphorylations of EGFR, p65, and ERK in eight colorectal cancer tissues.
Fig 2.
Go analysis of significantly regulated phosphoproteins.
(A) Workflow of the analysis of PRL-3’s impact on protein phosphorylation and cytokine secretion in HCT116 cells by antibody array. (B) PRL-3 induced phosphoproteome profile. Total number of phosphoproteins, and percent of phosphorylated Ser (S), Thr (T), and Tyr (Y) in the total significantly regulated phosphoproteins. (C) Functional characterization of the significantly regulated phosphoproteins, including biological processes, cellular components and molecular functions.
Table 1.
The top 20 phosphoproteins experiencing changes in phosphorylation in the PRL-3 overexpressing cells.
Fig 3.
Identification of significant pathways regulated by PRL-3.
(A) Significant KEGG pathways identified by DAVID when PRL-3 was overexpressed in HCT116 cells. The left-side ordinate values are -lg (Benjamini-Hochberg p-value), while the right-side ordinate values are the ratios of phosphorylated proteins in the total significantly regulated phosphoproteins. (B) Heatmaps of several pathways to show the phosphoproteins regulated by PRL-3, including ErbB signaling pathway, Focal adhesion, MAPK signaling pathway and Chemokine signaling pathway. The red color represents the phosphorylation of the protein was increased in PRL-3 overexpressing cells, while the blue color represents the phosphorylation of the protein was decreased in PRL-3 overexpressing cells.
Fig 4.
Significantly regulated cytokines by PRL-3.
(A) Significantly regulated cytokines were all increased in PRL-3 overexpressing HCT116 cells. (B) The mRNA levels of several regulated cytokines were increased in PRL-3 overexpressing HCT116 and LoVo cells. (C) Relative levels of GDF-15, IL-1α and NPY in the supernatants of PRL-3 overexpressing HCT116 and LoVo cells and corresponding control cells. The values are the mean and standard deviation, *p <0.05, **p <0.01; n = 3.
Fig 5.
IL-1α participates in PRL-3 induced cell migration.
(A) and (B) IL-1α was regulated by PRL-3 through NF-κB and Jak2-STAT3 signaling pathways. (A) The treatment of HCT116 cells with 10 μM BAY11-7082 for 24 hours reduced PRL-3-increased p-p65 (S536), and the treatment of HCT116 cells with 2 μM AG490 for 24 hours reduced PRL-3-increased p-STAT3 (Y705) while the treatment of HCT116 cells with 100 ng/mL IL-6 for 24 hours increased p-p65 (S536) and p-STAT3 (Y705). (B) The treatment of HCT116 cells with 10 μM BAY11-7082 or 2 μM AG490 for 24 hours reduced PRL-3-increased IL-1α, and the treatment of HCT116 cells with 100 ng/mL IL-6 for 24 hours enhanced PRL-3-induced IL-1α secretion. (C) The treatment of HCT116 cells with 5 ng/mL IL-1RN for 48 hours decreased PRL-3-induced HCT116 cell migration. (D) Inhibition of IL-1α by siRNA interference decreased PRL-3-induced HCT116 cell migration. The values are the mean and standard deviation, * p < 0.05, ** p < 0.01, *** p < 0.001; n = 3.