Supplementary Data:
RAPD finger printing
RAPD profiling of all the 10 Lactobacillus strains was generated using Oligo primer 228 (gct ggg ccg a),
244 (cag cca acc g) and 275 (ccg ggc aag c). PCR reaction was performed in a 25µl reaction volume containing PCR
Buffer (2.5µl), Oligo Primer (2.0µl), 200 µM dNTP (2.0µl), 1.0 U Taq DNA polymerase (0.5µl) and template
(2.0µl). The PCR cycling parameters included an initial denaturation of 95°C/ 5 min, followed by 45 cycles each of
denaturation (95°C/ 30 sec), annealing (42°C/ 30 sec), extension (72°C/ 2min) and final extension of 72°C/ 10 min.
The PCR products were electrophoresed on 1.8 – 2 % agarose gel with ethidium bromide (0.5 µg/ml) (Amersham
Biosciences, USA) at 100 V using 1 X TAE buffer. The amplified PCR products represented on the gel were
visualized under UV transilluminator and photographed with the help of gel documentation system (G-Box,
Syngene).
RAPD profiles of colonies isolated from faecal samples
Faecal samples were collected every alternate day of probiotic administration / PBS (Control group). Faecal
material (100 mg) was homogenized in 1ml of PBS. Serial dilutions were made and plating for lactobacillus counts
was done on MRS -BCP agar. The plates were incubated at 37°C for 24-48 hrs. Colonies were counted and
recorded. Colonies from different faecal count plates were randomly picked up and grown for 16 to 18 hrs at 37 oC
after inoculation into MRS broth. The genomic DNA from faecal cultures was then isolated by Pospiech and
Neumann (1995) method. PCR with random RAPD primers was carried out as described above. The RAPD patterns
were compared with the patterns of the pure cultures of the administered probiotic strain.
Primer Design
The primers for the house keeping genes (β-Actin for THP-1 cells and ACTB for mouse aorta) and target
genes (TNF-α and IL-6 from THP-1 cells and TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E selectin from mouse
aorta) were either taken from the published papers or designed from the genome sequence available at NCBI gene
bank and using Primer 3 plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). The
primers were got custom-synthesized from Imperial Biomed (Integrated DNA Technologies, Inc.; www.
idtdna.com) and Sigma, USA. While designing the primers for Real Time PCR, the G-C content was kept within 40-
60%, the amplified products size in the range of 100-200 bp and Tm in between 55-60oC. The specificity of the
designed primer pair was ensured by BLAST analysis. The amplified products were checked on 2% agarose gel
followed by melt curve analysis of the PCR amplified products to rule out the possibility of primer dimer formation.
Relative expression software tool (REST 2009)
Relative expression software tool (REST 2009) (www.genequantification.info), are generally used in
application for group wise comparison and statistical analysis of relative expression results in qRT-PCR. The
randomization test method, as a part of REST software, was used to assess statistical significance of up or down
regulation of target genes after normalization to the reference gene. The randomization test randomly reallocates the
observed values in different sample groups to the values in the control group. For up-regulation, the factor of
regulation is equal to the given value in the Randomization Data Output Box and for down-regulation, the factor of
regulation is illustrated as a reciprocal values (1/expression ratio) (Pfaffl 2002). Randomization test randomly
reallocates the observed values in different sample groups to the values in the control group. Relative expression is
then estimated for each of the new groups and the effect of randomization in each group is evaluated. If this effect is
not significant, it provides sufficient evidence that the observed treatment effect was not due to the random
allocation. Statistical analysis was considered significant at P ≤ 0.05. Data that emerged from this study has been
presented in the form of whisker-box plots and bar diagrams. The top and bottom of each box represent the 25th- to
75th-percentile values while the 50th percentile (median) is the horizontal dotted line in the rectangle. The vertical
bars represent the minimum and maximum values that are not considered outliers.
Figure: 1S - In vivo Experimental Design
Experimental Design of LPS Induced mouse model
Figure: 2S - Genus and Species specificity of Lactobacillus cultures by PCR
Genus (A) and Species (B) specificity of Lactobacillus cultures by PCR
Fig. 4.1 Genus (A) and Species (B) specificity of Lactobacillus cultures by PCR
Fig. 3S - RAPD profiles of selected Lactobacilli cultures with 228, 244 and 275 random primers
Figure: 4S - Confirmation of specificity of primers for ‘TNF-α’, ‘IL-6’ and ‘β-Actin’
Specificity of RT–qPCR products generated from the amplification of β-actin (reference) and TNF-α and
IL-6 (target) genes using specific primers was checked on agarose gels. A specific single product of the desired
amplicon i.e. 238 bp (β-actin), 134 bp (TNF-α) and 349 bp (IL-6) was obtained as revealed on 2% agarose gels. In
order to further assess the specificity of the amplified PCR products, melt curve analysis was also performed which
resulted in a single product of specific melting temperatures of 89.5oC, 89.6oC and 87.72oC for β–actin, TNF-α and
IL-6 respectively.
Figure: 5S - Standard curves for target (TNF- α and IL-6) and reference genes (β-Actin)
RT-qPCR efficiencies and linearity were calculated by preparing standard curves for the target and
reference genes from the given slopes plotted with the help of LightCycler software. C q values versus log cDNA
(reverse transcribed total RNA) concentration inputs by preparing serial dilutions of cDNA i.e. 300, 200, 100, 50
and 25 ng respectively were plotted to calculate the slope (mean ± SD; n = 3). The corresponding real-time PCR
efficiency (E) of one cycle in the exponential phase in respect of each gene was calculated according to the equation:
E = 10[–1/slope]. As is quite evident from the figure, the curves showed high linearity (Pearson correlation coefficient
R2 > 0.98). The slopes of β-Actin, TNF-α and IL-6 curves were -3.470, -3.730 and -3.568 respectively that indicated
high real-time PCR efficiencies of 1.94, 1.86 and 1.91 respectively.
Figure 5S: Whisker-box plots showing relative expression of TNF-α and IL-6 in THP-1 cells on challenge with
different strains of probiotic lactobacilli and LPS stimulation
Boxes represent the interquartile range, or the middle 50% of observations. The dotted line represents the median
gene expression. Whisker-box plots represent the minimum and maximum observations.
Figure: 7S - Specificity of primers for ACTB, TNF-α, IL-6, MCP-1, VCAM-1 , ICAM-1 and E selectin genes
by RT-qPCR based on electrophoretic separation and melt curve analysis
Specificity of the primers against the target genes viz. TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E
selectin along with ACTB as the reference gene was checked by amplification of the desired RT-qPCR products.
The specific amplicons of desired sizes specific for the target genes such as 112 bp (ACTB), 212 bp (TNF-α), 209
bp (IL-6), 96 bp (MCP-1), 86bp (VCAM-1), 82 bp (ICAM-1) and 85 bp (E selectin) were detected on agarose gels
after electrophoresis and also resulted in a single product of specific melting temperatures of 83, 88.6 79.5, 86.5, 85,
84.4 and 84oC for ACTB, TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E selectin respectively by melt curve
analysis.
Figure: 8S - Standard curves for the target and reference gene
RT-qPCR efficiencies and linearity were calculated by preparing standard curves for the target genes along
with reference gene from the given slopes plotted with the help of LightCycler software. Cq values versus log cDNA
(reverse transcribed total RNA) concentration inputs by preparing serial dilutions of cDNA were plotted to calculate
the slope (mean ± SD; n = 3). The corresponding real-time PCR efficiency (E) of one cycle in the exponential phase
in respect of each gene was calculated according to the equation: E = 10[–1/slope] (Rasmussen, 2001). All curves
showed high linearity (Pearson correlation coefficient R2 > 0.955). The values obtained in respect of slopes of
ACTB, TNF-α, IL-6, MCP-1, VCAM-1, ICAM-1 and E selectin were -3.3817, -3.3769, -3.369, -3.4432, -3.328 and
-3.387 respectively that indicated high real-time PCR efficiencies ranging from 1.95- 1.99.
Figure 9S: Whisker-box plots showing relative expression of TNF-α and IL-6 in LPS induced mouse model
Boxes represent the interquartile range, or the middle 50% of observations. The dotted line represents the median
gene expression. Whisker-box plots represent the minimum and maximum observations.
Figure 10S: Whisker –box plots in respect of Relative expression of MCP-1, VCAM-1, ICAM-1 and E selectin
in mice model.
Boxes represent the interquartile range, or the middle 50% of observations. The dotted line represents the median
gene expression. Whisker-box plots represent the minimum and maximum observations.
Figure: 11S - RAPD profiles of faecal isolates
RAPD profiles of faecal isolates
RAPD based DNA fingerprinting technique was used as a tool for identification of faecal isolates picked
from the faecal samples of experimental animals. Around 20 colonies were picked randomly and used for RAPD
profiling using primer 275. The typical banding patterns of the faecal isolates were compared with Lp91 as the test
strain (Fig. 12S). From the banding patterns, it is quite clear that the faecal isolates exhibited remarkable similarity
with the RAPD profile of the pure strain of Lp91, thereby, establishing their true identity as Lp91 in the mice gut
flora.