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SUPPLEMENTARY INFORMATIONS
Gut microbiota analysis
Denaturing Gradient Gel Electrophoresis (DGGE) was performed on the total bacteria
amplicons that were generated with primers targeting the V3 region of bacterial 16S rRNA
gene. The sequences are reported in Supplementary Table 1. DGGE on the total bacteria
amplicons was performed using the primers described by Muyzer et al. (Muyzer et al., 1993),
which amplify the V3 region of the bacterial 16S rRNA gene. The PCR mixture (50 µl)
contained 5 µl of 10x NH4 reaction buffer; 2.5 µl of MgCl2 (50 mM, 0.1 mg/ml); 2.5 µl of a
deoxynucleoside triphosphate preparation (containing each nucleotide at a 2 mM
concentration); 1 µl of each primer (10 µM); 0.25 µl of Taq polymerase (5 U/µl); 36.75 µl of
distilled water and 1 µl of DNA. This PCR program was used as follows: an initial
denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 20 s, annealing at 55°C
for 45 s, and extension at 72°C for 1 min; and a final extension at 72°C for 7 min, followed by
cooling to 4°C. The PCR was verified by mixing 10 µl of the PCR product with 2 µl of a
loading dye and electrophoresing it on a 1.5% (wt/vol) agarose gel for 15 min at 120 V
flanked by a 1 kb DNA ladder (Promega, Madison, WI).
The PCR products were analyzed on a DGGE gel using a protocol described by Muyzer et al.
(Muyzer et al., 1993) and modified as described below. The polyacrylamide gel (185 × 155 ×
1 mm) consisted of 8% (vol/vol) polyacrylamide in 1x TAE buffer. By diluting a 100%
denaturing polyacrylamide solution [containing 7 M urea and 40% (vol/vol) formamide] with
a polyacrylamide solution containing no denaturing components, polyacrylamide solutions of
35% and 60% denaturing compounds were obtained. The denaturing gel was allowed to
polymerize for 3 h, after which a 2.5-ml nondenaturing stacking gel containing a 30-well
comb was poured on top. After 1 h of polymerization, the PCR samples were loaded into the
wells. Electrophoresis was performed for 14 h at 85 V in 1x TAE buffer at a constant 60°C
temperature using a Dcode system (Model DGGE-2001, CBS Scientific Company). The gel
was stained with SYBR Gold (20 µl of SYBR Gold in 250 ml of 1x TAE buffer) for 30 min;
this was followed by examining the DGGE band profiles under a UV light. Digital image
capturing was performed using a UVtec UVIdoc gel documentation system. The obtained
DGGE fingerprints were analyzed using FPQuest software (Bio-Rad, Hercules, CA).
To detect the total bacteria and Lactobacillus spp., quantitative PCR was performed as
described for mRNA analysis. To detect Bifidobacterium spp. and Bacteroides spp., we used
probes and TaqMan Universal PCR Master Mix as described previously (Delroisse et al.,
2008). The primers and probes are detailed in Supplementary Table 1. The cycle threshold of
each sample was compared to a standard curve (performed in triplicate) made by diluting the
genomic DNA (5-fold serial dilution) (BCCM/LMG, Ghent, Belgium and DSMZ,
Braunshweig). Prior to isolating the DNA, the cell counts were determined in culture and
expressed as “colony-forming units” (CFUs).
Cell proliferation (MTT, manual counting, BrdU)
The cell growth assay is based on metabolically active cells cleaving yellow thiazolyl blue
tetrazolium bromide (MTT) to form purple formazan crystals. In a 96-well plate, cells were
incubated with different chemicals for the times indicated in the figure legends. MTT was
added to each well. After centrifugation, half of the medium was removed, and the formazan
crystals were dissolved by the addition of acid isopropanol. The formazan absorbance was
measured at 570 nm, from which a background value, measured at 650 nm, was subtracted.
Cell proliferation is expressed as a percentage of the value obtained for cells incubated with
the vehicle (medium, PBS or DMSO, final concentration between 0.1 and 0.2%). For manual
counting, cells were incubated in a 6-well plate with different chemicals. At each time point,
cells were harvested, centrifuged and suspended in PBS. Intact cells (determined by exclusion
of erythrosine) were counted under a microscope in a Bürker cell. The bromodeoxyuridine
incorporation assay was performed following the manufacturer’s instructions (Cell
Proliferation ELISA, BrdU colorimetric, Roche, Mannheim, Germany). All of the assays were
performed in media containing 10% fetal bovine serum.
Reference List
Delroisse JM, Boulvin AL, Parmentier I, Dauphin RD, Vandenbol M, Portetelle D (2008)
Quantification of Bifidobacterium spp. and Lactobacillus spp. in rat fecal samples by realtime PCR. Microbiol Res 163 (6): 663-670
Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations
by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified
genes coding for 16S rRNA. Appl Environ Microbiol 59 (3): 695-700