FIGURE LEGENDS
Figure 1. In vitro anti-proliferative effect of tigecycline and 5-FU . (A) Cell proliferation evaluated by MTT assay (B) Clonogenic assay in HCT116, Caco-2 and NCM356 cell lines treated with different doses of tigecycline or 5-FU. (C) Immunofluorescence representative images of Ki67 (green) staining in HCT116 cells non-treated or treated with tigecycline or 5-FU, cells in proliferation based on ki-67 positive cells and total cells (right and up) and ki-67 mean fluorescence intensity (MFI) (right and down). Scale bar: 50 μm. Data are presented as mean ± SEM. *P < 0.05, **P < .01, ***P < .001 vs. control.
Figure 2 . Molecular mechanisms involved in the anti-proliferative effect of tigecycline and 5-FU : effect on Wnt/β-catenin pathway. (A) Impact on β-catenin nuclear and cytoplasmic levels analyzed by Western blot in basal conditions or (B) stimulated with Wnt3a (C) Effect on the expression of Wnt/β-catenin target genes in the presence or absence of Wnt. Data are presented as mean ± SEM. *P < 0.05, **P < .01, ***P < .001, ****P<.0001 vs. control. #P < 0.05, ##P < .01, ###P < .001, ####P<.0001 vs. control + Wnt.
Figure 3 . Pro-apoptotic effect of tigecycline and 5-FU in vitro and molecular mechanisms involved in this effect. (A) Apoptosis study by Annexin-IP assay in HCT116 cell line. (B) Tunel assay in HCT116 cell. Scale bar: 100 μm (C) Impact on extrinsic apoptosis pathway (D) intrinsic apoptosis pathway (E) and endoplasmic reticulum mediated apoptosis pathway. Data are presented as mean ± SEM. *P < 0.05, **P < .01, ***P < .001, ****P<.0001 vs. control.
Figure 4 . Impact of tigecycline and 5-FU on tumorigenesis in CAC mice (n=10) . (A) Representative colonoscopy images and tumor score based on the size of the tumor evaluated by colonoscopy (B) Representative colon images of CAC, CAC-T25, CAC-T50 and CAC-FU mice, (C) tumor number and (D) tumor size. (E) Representative images of histological sections of colon. Adenocarcinomas are indicated with red arrows, adenomas with blue arrows and a lymph node with black arrow. Scale bar: 500 μm. (F) Ki67 immunofluorescence staining (green) was conducted to identify actively proliferating cells and proliferation index was calculated from the percentage of ki67-positive cells in ratio to total cells per field. Scale bar: 50 μm (G) Protein levels and gene expression of several markers involved in cell proliferation evaluated and detected in colon. Data are presented as mean ± SEM. Bars with different letter differ statistically (P<0.05).
Figure 5 . Impact of tigecycline and 5-FU on stemness and apoptosis in CAC mice. (A) Representative flow cytometry analysis of LGR5+ population and quantification of LGR5+ and LGR5+CD44+ cells populations in each experimental group. (B) Changes in SNAI1 levels induced by the CAC process and tigecycline treatment analyzed by Western blot in colon. (C) Representative fluorescence images of Tunel assay-stained (apoptosis) colonic tissue sections from CAC, CAC-T25, CAC-T50 and CAC-FU mice (n=4). Apoptotic cells are stained in green and nuclei in blue (Hoechst). The apoptosis index was calculated as Tunel⁺ cells/total cells per field. Scale bar: 100 μm. (D) Levels of several markers involved in apoptosis and cell survival analyzed in colon tissue by Western blot. Data are presented as mean ± SEM. Bars with different letter differ statistically (P<0.05).
Figure 6 . Effect of tigecycline and 5-FU on CRC-associated inflammation and lymphocytes (CD3+) populations. (A) DAI score (n=10). (B) Impact on the cytokines gene expression quantified by real-time qPCR (n=8). (C) Representative dot plots from HEALTHY, CAC, CAC-T25, CAC-T50 and CAC-FU mice (n=4-5) showing the CD4 vs. CD8 T lymphocyte distribution. (D) CD3+CD4+, Th1, CD3+CD8+ and Tc1 populations levels in HEALTHY, CAC, CAC-T25, CAC-T50 and CAC-FU mice groups. Fold increase was calculated vs. control group. Data are presented as mean ± SEM. Bars with different letter differ statistically (P<0.05).
Figure 7. Effect of tigecycline and 5-FU on gut microbiota composition (n=9). (A) Microbiome diversity indexes calculated after faecal microbiota Illumina sequencing: observed species, Simpson index, Shannon index and ACE index. (B) β-diversity by principal coordinate analysis score plot. (C) A Venn diagram showing the number of OTUs which are unique and common to each experimental group. (D) Distribution histogram of relative abundance of taxa. (E)Bacillota/Bateroidota (named F/B) ratio in each experimental group (F) A heatmap showing the taxonomic signatures at the genus level. Data are presented as mean ± SEM. Bars with different letter differ statistically (P<0.05).
Figure 8 . Effect of tigecycline and 5-FU on gut microbiota composition and correlation between bacterial abundance and functional features in each group (n=9). (A) Volcano plot indicating the upregulated and downregulated OTUs in Healthy, CAC-T25, CAC-T50 and CAC-FU in comparison with those OTUs determined on CAC group. (B) Venn-diagrams that show the down-regulated shared OTUs and the up-regulated shared OTUs between Healthy, CAC-T25, CAC-T50 and CAC-FU when compared with CAC group. (C) Heat-map of correlations between gut microbiota changes and different features analyzed in vivo between tigecycline-treated mice and untreated-CAC mice.