Figure 1.
Correlation between bacterial metabolites production and AP1 activity.
A) Bacterial phyla distribution in healthy human and in our selected bacterial collection. Inner part of the cycle represents the phyla distribution in our collection, outer part was based on the latest data from Tap et al. [60]. B) PCA showing the correlation between bacterial phyla, acid production and activation of AP-1 pathway. A positive correlation is observed for Firmicutes (violet) and Fusobacteria (pink). Bacteroidetes (orange), Actinobacteria (green). C) A detailed analysis using a three dimensional plot confirms the strong positive correlation between Butyrate (violet circles) producing bacteria and induction of AP-1 response and shows that no positive correlation is observed for propionate producing bacteria (orange circles) and non butyrate/propionate producing bacteria (black circles). Bacteria producing both butyrate and propionate are presented with yellow circles. AP-1 response (log RLU), propionate and butyrate concentration in (mM).
Figure 2.
Dose-response of SCFA on AP-1 pathway activation.
HT-29/AP-1 cells were exposed to increasing concentrations for 24 h. Data are mean ± standard error of the mean (SEM) of triplicate measurement of one representative of three independent experiments; ***P<0.001, **P<0.005, *P<0.05 as compared to control.
Figure 3.
Activation of AP-1 pathway following HT-29 cell treatment with different SCFAs: acetate (8 mM), propionate (4 mM), butyrate (2 mM); or TSA (500 nM) and concomitantly treated with PMA (0.1 µM) (A) or EGF 10 ng/ml B). Dose response of PMA C) or EGF D) in the presence of butyrate (2 mM) or TSA (500 nM). Similar synergy upon association of PMA and butyrate is seen in another AP-1 reporter cell line Caco2/AP-1 E) Effect of bile acids: DCA (10 µM), UDCA (10 µM) associated or not with Butyrate (2 mM) (F). Data are mean ± standard error of the mean (SEM) of triplicate measurement of a representative of three independent experiments ***P<0.001, **P<0.005, *P<0.05 as compared with the control (Two way ANOVA); ### P<0.001, ## P<0.005, #P<0.05 as compared with the activator used (A, E : PMA ; B : EGF).
Figure 4.
c-Fos A) and Cyclin D1 B) gene expression determined by Quantitative real-time PCR on total RNA extracted from cells exposed to PMA (0.1 µM), Na-Butyrate (2 mM), or both of them, for 1 h and 6 h, respectively. A positive, synergistic effect is observed on c-Fos expression for cells co-stimulated with PMA/Na-butyrate, while, in the same conditions, an opposite effect is observed for cycline D1. Data are mean + standard error of the mean (SEM) of (triplicate) measurement of a representative of three independent experiments. Different letters indicate statistically different results (p<0.05).
Figure 5.
C-fos time course upon PMA induced activation.
Cells were treated with PMA (0,1 µM), butyrate (2 mM), TSA 500 nM, or association of PMA with butyrate or TSA for 0,5, 1 h A), 6 and 24 h B) and total proteins were Western blotted for c-fos. A synergistic effect following PMA/Butyrate co-stimulation is observed after 6 h of stimulation and 24 h. Western blot of GAPDH (lower panel) was shown as loading control. Data are representative of three independent experiments.
Figure 6.
Effect of several kinase inhibitors on PMA (1 µM), Butyrate (2 mM) and PMA/Butyrate stimulated luciferase activity of HT-29 reporter cells.
Bisindolylmaleimide (BIM 10 µM), UO126 (10 µM), SB203580 (10 µM), PD98059 (10 µM), H-89 (10 µM). Reporter gene activity was measured after 24 h stimulation. Results are mean + standard error of the mean (SEM) of triplicate measurements of a representative of three independent experiments ***P<0.001, **P<0.005, *P<0.05 compared with the control and ### P<0.001, ## P<0.005, #P<0.05. P values were determined by the t-test.
Figure 7.
PMA induced activation of MAPK kinase pathway.
Cells were treated with PMA (0.1 µM), butyrate (2 mM) or both of them for 30 min and total proteins were Western blotted for pp38, pERK1/2 and pJNK. A synergistic effect following PMA/Butyrate co-stimulation is observed in the case of ERK. Western blot of β-actin (lower panel) was shown as loading control. Data are representative of three independent experiments.
Figure 8.
Effect of butyrate and on cell proliferation of Caco-2 A), HT-29 B) and on alkaline phosphatase activity as marker of differentiation in Caco-2 cells C). Data are representative of three independent experiments. Different letters indicate statistically different results (p<0.05).