Hierarchical clustering of genes involved in NF-κB activation (A) and ECM remodeling (B).

<p>Expression levels of each gene in the various treatment groups relative to the unstimulated control group are displayed as fold changes. Red or green color indicates the fold change of up- or down-regulated expression levels, respectively, compared with unchanged expression shown in black. BCM, <i>Bifidobacterium infantis</i>-conditioned media; LCM, <i>Lactobacillus acidophilus</i>-conditioned media.</p

GeneGo MetaCore gene enrichment analysis of upregulated genes in H4 cells.

<p>The top 10 pathways (A) and networks (B) differentially affected between the negative control and probiotic-conditioned media treatments under IL-1β stimulation were obtained from the analysis. BCM, <i>Bifidobacterium infantis</i>-conditioned media; LCM, <i>Lactobacillus acidophilus</i>-conditioned media. NF-kB, nuclear factor-kappa B; PDE4, cAMP-specific phosphodiesterase-4 family protein group; PEDF, pigment epithelium-derived factor protein; GnRH, gonadotropin-releasing hormone; APRIL, tumor necrosis factor (ligand) superfamily members 13; BAFF, tumor necrosis factor (ligand) superfamily members 13b; CF, cystic fibrosis; MIF, macrophage migration inhibitory factor.</p

The mRNA and protein expression of IL-6 and IL-8 in H4 cells.

<p>Gene expression was determined by qRT-PCR (A) and protein level was measured by ELISA (B). Expression levels of genes are normalized to glyceraldehyde-3-phosphate dehydrogenase (GADPH). Relative mRNA levels were calculated using the 2^-ΔΔCt method and the average ΔCt values of the unstimulated control group served as the calibrator. The gene expression and cytokine production in probiotic-conditioned media treatments was compared to the corresponding control group, unstimulated and IL-1β-stimulated, respectively. All data represent the mean ± the SEM (n = 3 for qRT-PCR and n = 4 for ELISA). A p<0.05 (*) or p<0.001 (**) depicts the significance value. BCM, <i>Bifidobacterium infantis</i>-conditioned media; LCM, <i>Lactobacillus acidophilus</i>-conditioned media.</p

Degradation of cytoplasmic IκBα and nuclear translocation of NF-κB p65 in H4 cells.

<p>Western blot was performed and densitometry of immune blot bands was used for quantification. The protein levels of cytoplasmic IκBα, as well as cytoplasmic and nuclear NF-κBp 65 (A) and quantification of each immuno blot band (B) are displayed. The protein levels in probiotic-conditioned media treatments were compared to the corresponding control group, unstimulated and IL-1β-stimulated, respectively. All data represent the mean ± the SEM (n = 3). A p<0.05 (*) or p<0.001 (**) depicts the significance value. Immunofluorescence staining of NF-κB p65 (green) was performed in H4 cells (C). The fields presented were randomly captured to accurately represent each condition. BCM, <i>Bifidobacterium infantis</i>-conditioned media; LCM, <i>Lactobacillus acidophilus</i>-conditioned media.</p

Validation of microarray results of selected genes in H4 cells by qRT-PCR.

<p>The RNA used for qRT-PCR was from the same samples used for microarray analysis. NF-κB activation (A) and ECM remodeling (B) associated genes with significant changes are shown. Expression levels of genes are normalized to glyceraldehyde-3-phosphate dehydrogenase (GADPH). Relative mRNA levels were calculated using the 2^-ΔΔCt method and the average ΔCt values of the unstimulated control group served as the calibrator. The gene expression levels of probiotic-conditioned media treatments were compared to the corresponding control group, unstimulated and IL-1β-stimulated, respectively. The data represent the mean ± the SEM (n = 3). A p<0.05 (*) or p<0.001 (**) depicts the significance value. BCM, <i>Bifidobacterium infantis</i>-conditioned media; LCM, <i>Lactobacillus acidophilus</i>-conditioned media.</p

Premature gut microbiota variation in the first two month of life.

<p>The gut microbiota variation at class level over 0–60 days of life from all the samples is visualized by NMDS plot. The color gradient from red to blue represents the day of life of the babies. The microbiota from early day of life is distinguished from the elder days.</p

NMDS of early and late onset NEC and controls at the genus level.

<p>The difference between NEC and controls is displayed for early onset NEC (Fig. 2A) and late onset NEC (Fig. 2B) with their controls by NMDS plot. Each dot represents one sample. Green dots represent the controls and red dots represent the NEC samples. Early NEC subjects and control subjects have a clear separation at second week of the life. The distinction between late onset NEC and controls is less obvious except at the third week of life.</p

Microbiota progression before early onset NEC and late onset NEC at class and genus level.

<p>The relative abundances of four most dominant classes (Fig. 3A-early onset at class level, 3B-late onset at class level) and the genera (Fig. 3C-early onset at genus level, 3D-late onset at genus level) that are significantly different between NEC and controls are plotted at 7–9 days, 4–6 days and 1–3 days prior to NEC onset. In early onset NEC category, 3,6, 8 NEC samples were included at 7–9 days, 4–6 days and 1–3 days; 8, 5, 6 control samples were included in the above time points. In late onset NEC category, 7, 7,10 NEC samples and 6, 8, 4 control samples were included in the above time points. Red and green of the boxplots indicate NEC and control samples, respectively. The asterisks indicate the significant difference between NEC and control.</p

PSA reduced IL-1β induced P-c-Jun expression is TLR2 dependent.

<p>P-c-Jun (green color) was detected by immunofluorescent staining in control (A1), TLR2 knockdown (B1) and TLR4 knockdown (C1) H4 cells which were pretreated with or without PSA for 24 hrs and then exposed to IL-1β for 2 hrs The nuclei were counterstained by Dapi (blue), and the cell membrane was stained red. Amplified x600; scale bar = 20 μm; n = 60–70 cells/treatment. The images were representative of three separate experiments with similar results. The analysis (A2, B2 and C2) applied by corrected total cell fluorescence (CTCF) was determined.** P<0.01, ***P<0.001(one-way ANOVA and post-hoc tests).</p

PSA reduced IL-1βinduced IL-8 in H4 cells.

<p>H4 cells were pretreated with or without PSA at an optimal dose and then exposed to IL-1β or treated with PSA alone. The supernatants were collected to measure IL8 levels by Elisa. The mean and SEM were from triplicate wells and were representative of three separate experiments with similar results. **P<0.01 (one-way ANOVA and post-hoc tests).</p