Fig 1: Knockdown of MUC2, GALNT7 or FUT2 decreased the adhesion of C. butyricum to HT-29 cells. n=3. (A) Knockdown efficiencies of MUC2, GALNT7 and FUT2 were examined by RT-qPCR in HT29 cells, and then western blot analysis was performed to verify the knockdown efficiencies. (B) After knockdown of MUC2, GALNT7 or FUT2, HT-29 cells were stained with FITC labeled agglutinin. The mean fluorescence intensity of each picture is 33.44, 31.25, 7.19, 5.76, 4.91 arbitrary units (AU) using ImageJ software. (C) Adhesion of C. butyricum to HT-29 cells was assayed by measure of the fluorescence intensity with the microplate reader at 528 nm. The si-MUC2-2, si-GALNT7-2 and si-FUT2-1 were used in this adhesion experiment. Results are expressed as means and standard deviation of three independent experiments with **P < 0.01 and *P < 0.05.
Fig 2: C. butyricum promoted the expression of GALNT7 and FUT2 of HT-29 cells. n=3. (A) Western blot analysis for GALNT7 and FUT2, normalized by β-actin. (B) Quantitative analysis for the densitometry of the proteins performed using ImageJ software. Results are expressed as means and standard deviation of three independent experiments with **P < 0.01.
Fig 3: Effects of FUT2 on the growth of SW-480-derived xenograft tumors in nude mice. (A) Images of mice and (B) tumors are presented. (C) The tumor volume of each group was measured and calculated using the formula described in the 'Materials and methods' section. (D) The tumors were weighed. *P<0.05 and **P<0.01. FUT2, fucosyltransferase 2.
Fig 4: FUT2 is overexpressed in colorectal cancer tissues and cell lines. (A) Boxplot illustrating the relative expression of FUT2 in normal, COAD and READ samples. (B) Evaluation of FUT2 expression in fresh human colorectal cancer tissues and adjacent normal tissues using western blot analysis. C, colorectal cancer tissues; N, normal tissues. (C) Representative images of the expression of FUT2 proteins assessed using immunohistochemistry. C1, colorectal cancer tissues; N1, adjacent tissues; CLNM, colorectal cancer lymph node metastatic tissue; NLN, normal lymph node tissue. Magnification: Upper panels, ×100; lower panels, ×400. (D) Quantitative analysis of the average MOD of FUT2 staining in normal tissues, colorectal cancer tissues and lymph node metastatic tissues. N, normal control; CI, cancer stage I; CII, cancer stage II; CIII, cancer stage III. (E) The expression of FUT2 in colorectal cancer cell lines and FHC was examined using western blot analysis. β-actin was used as the loading control. Data are expressed as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. FUT2, fucosyltransferase 2; COAD, colon adenocarcinoma; READ, rectal adenocarcinoma; MOD, mean optical density.
Fig 5: Effects of FUT2 on the migration and invasion of colorectal cancer cells. (A) The mRNA and (B) protein expression of FUT2 was examined using reverse transcription-quantitative PCR and western blot analysis, respectively. (C) The effect of FUT2 on cell migration was measured using a wound healing assay. The migration rates of SW-480 and DLD-1 cells were calculated using the formula described in the 'Materials and methods' section. Scale bar, 1 mm. (D) The effect of FUT2 on cell migration was measured using Transwell assay, and the numbers of migrated cells were calculated. Scale bar, 1 mm. (E) Matrigel invasion assay was performed to assess the invasion of SW-480 and DLD-1 cells, and the numbers of invasive cells were calculated. Scale bar, 1 mm. Data are expressed as the mean ± SD. *P<0.05, **P<0.01 and ***P<0.001. FUT2, fucosyltransferase 2.
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