Fig 2: APOA-1 and AIBP expression in normal and neoplastic intestinal tissues. a IHC staining for AIBP and APOA-I in normal intestinal tissues, small intestinal adenoma, large tumors and colon tumors in C57BL/6 J-ApcMin/+ mice; scale bar, 50 μm. b Western blot for APOA-1 and AIBP in intestinal normal, adenoma and tumor tissues in C57BL/6 J-ApcMin/+ mice. c AIBP or APOA-I mRNA expression in the patients’ normal and cancerous tissues was evaluated according to the tumor differentiation stage (n = 47; *P < 0.05; **P < 0.01; ***P < 0.0001). d Staining for AIBP and APOA-I in human normal and malignant colon tissues from patients; scale bar, 50 μm. e, f qPCR and western blot results showing AIBP or APOA-I mRNA expression in various cell lines, including normal and colon cancer cell lines with a varying degree of malignancy. N, normal epithelial cells; L, low-malignant colon cancer cells; H, high-malignant colon cancer cells (NS, no significant difference; *P < 0.05; **P < 0.01)

Fig 3: AIBP and APOA-I cooperate to inhibit tumor growth and metastasis. a–c Representative images and statistical analyses of tumors in mice inoculated with HCT116 (a) or SW620 cells (b) expressing the negative control (NC), AIBP, APOA-I and AIBP + APOA-I, and the average radiant efficiency [(p/s/cm2/sr]/(µW/cm2)] of tumors in mice (c) (n = 5; *P < 0.05; **P < 0.01). d The tumor growth rates were evaluated by calculating the tumor volume. e Representative tumors from mice injected with HCT116 cells expressing AIBP and subcutaneously administered APOA-I recombinant proteins (R-APOA-I, 0.5 mg/kg) at the end stage (mean ± SD, n = 5; *P < 0.05; **P < 0.01). f Representative picture and HE staining image after injection of the indicated cells. g Liver metastatic foci were quantified (mean ± SD, n = 8, **P < 0.01). h The survival rates of the indicated cell-transplanted mice (n = 6, 8, 8, and 8, respectively). Kaplan-Meier survival curves representing the percentages of live mice on the days indicated. Statistical significance among the studied groups was calculated using GraphPad Prism 6 software

Fig 4: Evaluation of the therapeutic effect of AIBP + APOA-I in ApcMin/+ mice. a Schematic representation of the recombinant protein treatment protocol as described in the Materials and Methods. b, c Representative tumor-bearing small intestines and colons were stained with 1% alcian blue, and the white arrows indicate the tumor nodules. d The total numbers of small intestinal and colorectal tumors per mouse were enumerated (Mean ± SD, *P < 0.05). e The proportion of tumors of different sizes in each group was calculated and analyzed (Mean ± SD, *P < 0.05, **P < 0.01)

Fig 5: The effect of Surf4 knockdown on the level of apoB and apoA-I in cultured cells. A–C: Knockdown of Surf4. Huh7 and HepG2 cells were transfected with scrambled (Scram.) or one of the two Surf4 siRNAs (Surf4-1 and Surf4-2). About 36 h after transfection, the cells were incubated with oleic acid for 4 h, washed, and then incubated in DMEM without FBS and oleic acid for 16 h. Whole cell lysate was prepared. Same amount of total proteins in whole cell lysate (top) and culture medium (bottom) was applied to Western blot using antibodies indicated. Similar results were obtained from at least five experiments. The relative densitometry was the ratio of the densitometry of target to that of transferrin receptor in whole cell lysate or albumin in medium in the same condition. The ratio of the relative densitometry was the ratio of the relative densitometry of the target in Surf4 knockdown cells to that of the same target in the control cells transfected with scrambled siRNA. The relative densitometry of the target in the control cells was defined as 1. D: Coimmunoprecipitation. The same amount of whole cell lysate from Huh7 or HepG2 was incubated with the preimmune serum (Pre) or a rabbit anti-Surf4 antibody (1195Sa) and protein-G agarose. Whole cell lysate (input) and immunoprecipitated proteins (IP-Beads) were applied to Western blot with antibodies indicated. Similar results were obtained from at least three more experiments. E–G: Confocal microscopy. Huh7 cells were fixed, permeabilized, and then incubated with a goat anti-apoB (abcam) and a rabbit anti-Surf4 antibody, followed by confocal microscopy; apoB (green), Surf4 (red), and DAPI (blue). An x–y optical section of the cells illustrates the cellular distribution of proteins (magnification: 325×). Representative images were shown.