Fig. 3

Genome-wide expression analysis identifies CD15/FUT4 as a novel RAF-MEK-ERK kinase downstream target. a The most enriched network of CD15/FUT4 regulators is depicted comprising 20 regulons and two closely connected upstream genes involving EGFR and FGFR pathways (see Additional file 2 and Additional file 3: Figure S5 for more information). The gene expression datasets GSE17536/GSE17537 series (n = 226) were analyzed simultaneously with the ARACNe algorithm to infer transcriptional regulatory network fromgenome-wide expression profiles CD15/FUT4-connected. b CD15/FUT4 expression in CRC using patient-matched tumor-normal data available from TCGA. The P value refers to Mann–Whitney test. Expression profiles of ERBB1, ERBB2, ERBB3, FGFR4, CD15/FUT4 and KRAS mutant across a series of CRC cell lines (n = 60) by applying a fold change of gene expression microarray data of 1.5 and subdivided on the basis of chromosomal instability calculated as fraction of copy-number alteration pattern (form 0 to 0.922). Significant correlation between CD15/FUT4, ERBB3 and FGFR4 expression levels. c Linear correlation between ERBB3, FGFR4 and CD15/FUT4 transcript levels is validated in our independent series of (n = 12) CRC cell lines. Immunofluorescence labeling “green” of nonpermeabilize cancer cells shows differential expression of CD15/FUT4 “arrows,” on the cell plasma membrane of RKO and HT29, respectively. To visualize nuclei, the merged images were stained with 4’6-diamidino-2-phenylindole (Dapi), “blue”. d CD15/FUT4-dependent transcript induction by EGF (10 nM) or IL1b (20U/ml) by using 0.1 % of DMSO as vehicle, in two representative CRC cell lines SW480 and RKO, respectively. Cells were treated for 8 h and the ratio anti-p-ERK/ERK1/2 was quantified by western-blot analysis. The treatment with IL-10 (200U/ml) or IL-6 (50 ng/ml) for 30 min revealed a significant induction of IL6-STAT1/STAT3 dependent phosphorylation in a panel of 4 CRC cell lines as detected by western-blot analysis. Mononuclear cells (Mono) purified from buffy coats of healthy donors were used as positive control for IL10-STAT3 dependent phosphorylation. CD15/FUT4 transcript did not reveal any significant induction following IL10 or IL6 at 48 h of treatment. The P value were obtained by Mann–Whitney test; *P ≤ .05; **P ≤ .01