Hierarchical clustering modulated genes during infection of Caco-2 cells with Shigella, in the presence or absence of L. casei. Caco-2 cells were incubated overnight in the presence or absence of L. casei and challenged for 3 h with M90T-AfaE, the virulent strain of S. flexneri. Radioactive nucleotides were incorporated during cDNA synthesis. The labeled probes were hybridized onto macroarrays consisting of 1050 human genes represented by PCR products spotted onto nylon membranes. After washings and scanning, signal detection and normalization were performed allowing group comparisons. Modulated genes were then clustered using dChip software. Each row represents a gene, and each column represents the mean expression for each replicate. The red color represents an expression level above the mean expression of a gene across all samples, and the blue color represents an expression level lower than the mean. For each condition, eight biological replicates were made.
Anti-inflammatory effect of L. casei. The cells were treated as described in Material and Methods and in the legend to Fig. 1. After RNA extraction, cDNA was synthetized using Oligo-dT and reverse transcriptase enzyme. Five microliters of a 1/20 dilution were used as a template for the PCR. A, Confluent, polarized Caco-2 cells were used. B, Densitometric quantification of PCR products ((gene/GAPDH) × 100).
L. casei infection results in up-regulation of I-κBα protein and inhibits Shigella-induced I-κBα degradation. A, Overnight preincubation of HEK293 cells with L. casei induces the up-regulation of I-κBα protein, while leaving other proteins of the Nod1-dependent signaling pathway unchanged. B, Overnight (O/N) preincubation of HEK293 cells with L. casei blocks I-κBα degradation induced by the invasive bacteria S. flexneri. CTR, Control.
L. casei infection results in up-regulation of the phosphorylated form of the I-κBα protein. Overnight (O/N) preincubation of HEK293 cells with L. casei (ranging from 2.5 106 to 5.107 bacteria/ml) induced a dose-dependent increase of I-κBα protein phosphorylation as determined by Western blotting using a specific polyclonal anti-human phospho-I-κBα Ab. CTR, Control.
Inhibition of TNF-α-induced NF-κB activation by L. casei. A, HEK293 cells were preincubated overnight with L. casei or MRS buffer (control medium) before stimulation for 4 h with TNF-α. Three bacterial concentrations were used: 5.106, 2.107, or 5.107 bacteria/ml. B, HEK293 cells were preincubated overnight with L. casei or MRS buffer (control medium) before stimulation for various times (15, 30, or 45 min) with TNF-α, followed by lysis of the cells. The I-κBα protein content was determined by Western blotting using a polyclonal anti-human I-κBα Ab. CTR, Control.
L. casei induced a down-regulation of genes involved in ubiquitination/degradation processes. Caco-2 cells were cultured overnight with L. casei with a MOI of 100. A, After washes with PBS and RNA extraction with the Rneasy mini kit, cDNA was synthetized using oligo-d(T) and reverse transcriptase. PCRs were performed as described in the legend to Fig. 2. B, After washes with PBS and lysis with Laemmli buffer, aliquots of the lysates were loaded on 15 or 10% SDS-PAGE for the subsequent detection of Rbx-1 or tubulin, respectively. The Rbx-1 or tubulin protein contents were determined by Western blotting using a polyclonal anti-human Rbx-1 or a monoclonal anti-human tubulin Ab, respectively. C, HEK cells were treated overnight with L. casei or for 6 h with 50 μM of the proteasome inhibitor MG-132 before incubation with or without TNF. The I-κBα protein content and global ubiquitinated proteins were determined by Western blotting (WB) using a polyclonal anti-human I-κBα Ab or monoclonal anti-ubiquitin Ab, respectively.
a Signal intensity resulting from macroarray hybridizations done with eight biological replicates of M90T infected Caco-2 cells were normalized and log2 transformed. Data analysis using the unpaired Welch t test from the dChip software gives a fold change between experimental point and baseline in a linear scale and an associated p value. Results with a p < 0.05 were considered as statistically significant.
Determination of modulated probe sets during time course culture of Caco-2 cells with L. caseia
Probe Set Number
a Caco-2 cells were cultured 2, 6, or 24 h with L. casei. After RNA extraction, labeling and hybridizations onto U133A Affymetrix GeneChip were performed. The number represents the modulated probe set with a fold change > 2.