Fig 2: LTA4H and BLT1 are overexpressed in human colon adenocarcinoma tissues and colitis and are associated with CRC patient survival probability. (a) LTA4H levels in human colon tissue were analyzed by immunohistochemistry and the density score from each sample was determined (right panel). Representative cases are shown (left panels). The asterisk (*, ***) indicates a significant (p < .05, p < .001, respectively) increase in LTA4H expression in colon adenocarcinoma tissue compared with normal tissue (scale bar, 100 µm). (b) LTB4 concentration in human serum was detected by ELISA assay. The asterisks (**) indicate a significant (p < .01) difference in CRC patients compared with normal adult subjects. (c) LTA4H levels in human colitis were analyzed by immunohistochemistry. The asterisks (***) indicate a significant (p < .001) increase in LTA4H expression in colitis tissue compared with normal tissue (scale bar, 100 µm). (d) Kaplan-Meier survival curves showed the life span of CRC patients with LTA4H high expression (n = 32) or LTA4H low expression (n = 31). (e) BLT1 levels in human colon tissue were analyzed by immunohistochemistry. The asterisks (**, ***) indicate a significant (p < .01, p < .001, respectively) increase in BLT1 expression in colon adenocarcinoma tissue compared with normal tissue (scale bar, 100 µm). (f) BLT1 levels in human colitis were analyzed by immunohistochemistry. The asterisks (***) indicate a significant (p < .001) increase in BLT1 expression in colitis tissue compared with normal tissue (scale bar, 100 µm). (g) Kaplan-Meier survival curves relative to BLT1 expression were analyzed for CRC patients.

Fig 3: LTA4H mediates cell transformation. (a) Knockdown of LTA4H expression in DLD-1 and HCT-15 colon cancer cell lines. (b) LTB4 production is decreased in DLD-1 and HCT-15 colon cancer cell lines after knockdown of LTA4H. The data are shown as mean values ± S.E. from triplicate experiments. The asterisk (*) indicates a significant (p < .05) decrease in LTB4 production in shLTA4H cells compared to shMOCK cells. (c) DLD-1 or HCT-15 cells were transfected with shLTA4H or shMOCK and grown in soft agar and then colonies were counted. The data are shown as mean values ± S.E. from triplicate experiments. The asterisk (*) indicates a significant (p < .05) decrease in colony formation in shLTA4H cells compared to shMOCK cells. (d) DLD-1 or HCT-15 colon cancer cells were transfected with shLTA4H or shMOCK. Cells were harvested and the expression levels of phosphorylated and total proteins were analyzed by Western blotting.

Fig 4: mRNA expression of 5-LO (A), LTA4H (B), LTC4S (C), PTGS2 (D), PGES (E), PGFS (F), and PGIS (G) in uterine tissues (endometrium and myometrium) on 4th and 13th day of the estrous cycle, and in pregnancy and anestrus phase. Data were normalized against GAPDH for mRNA expression using GraphPad PRISM (Version 8.3.0). Each bar represents one experimental group with SEM. Statistical differences were analyzed withy two-way analysis (ANOVA) of variance followed by Tukey’s post hoc test. The lowest statistical significance was p < 0.05. Asterisks indicate statistical differences between endometrium and myometrium (* p < 0.05; ** p < 0.01; *** p < 0.001). Different letters indicate statistical differences (p < 0.05) between the experimental reproductive stages throughout endometrium (a, b) and myometrium (A, B), respectively.

Fig 5: Protein expression of 5-LO (A), LTA4H (B), LTC4S (C), PTGS2 (D), PGES (E), PGFS (F), and PGIS (G) in uterine tissues (endometrium and myometrium) on 4th and 13th day of the estrous cycle, and in pregnancy and anestrus phase. Data were normalized against ACTB for protein expression using GraphPad PRISM (Version 8.3.0). Each bar represents one experimental group with SEM. Statistical differences were analyzed with two-way analysis (ANOVA) of variance followed by Tukey’s post hoc test. The lowest statistical significance was p < 0.05. Asterisks indicate statistical differences between endometrium and myometrium (* p < 0.1; *** p < 0.001). Different letters indicate statistical differences (p < 0.05) between the experimental reproductive stages throughout endometrium (a, b) and myometrium (A, B), respectively.