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.