S. aureus (ATCC29213) was purchased from Micro biologics Inc. (St. cloud, MN, US). Mouse IL-1α, IL-1β and TNF-α enzyme-linked immunosorbent assay (ELISA) kit was purchased from Cusabio Biotech (Wuhan, Hubei, China). Trizol was purchased from Invitrogen (Carlsbad, CA, US). The reverse transcriptase synthesis kit was purchased from Takara Biotech (Dalian, Liaoning, China). SYBR Premix Ex TaqTM II was purchased from Sheng gong Biotech (Shanghai, China).
Eighty BALB/c female mice and ten male mice (60 days old) were purchased from the Center of Experimental Animals of Hebei Agricultural University and the protocol was approved by the Animal Care and Ethics Committee of the Hebei Agricultural University. The mice were fed separately in an air-conditioned room maintained at a temperature of 24 ± 1°C for 2 weeks to adapt to the environment. The diet was prepared consulting the standard of AIN-93 purified diets for laboratory rodent in Animal Nutrition and Feed Science Laboratory of Hebei Agricultural University. Pregnancy is determined by vaginal plugs. Pregnant mouse was randomly divided into four groups: Control group and Experimental group. Control group (CG; Se-deficient basal diet during pregnancy without S. aureus infection; n=20), Negative control (NG; 0.2 mg/kg Se-supplemented diet during pregnancy without S. aureus infection; n=20), Positive group (PG; Se-deficient basal diet during pregnancy and infected with S. aureus four days after pregnancy; n=20) and Treatment group (TG; 0.2 mg/kg Se-supplemented diet during pregnancy and infected with S. aureus four days after pregnancy; n=20). 24 h after infection, all mice were euthanized and the mammary tissues were sampling.
The tissues were collected and fixed in 10% formaldehyde solution, and then embedded in paraffin wax. The mammary tissue sections were stained with hematoxylin eosin (H&E) staining and the pathological changes were observed under a microscope (Olympus DX45, Japan).
Scanning electron microscope (TEM) examination
Mammary gland was perfusion fixed with 2.5% glutaraldehyde and 2% formaldehyde containing 2 mM CaCl2 in 0.025 M sodium cacodylate buffer (pH 7.3) at 35 °C for 10 minutes. Tissues were then removed and placed in the same fixative for 2–3 hours at 4 °C. First, semi-thin slides were made using an ultramicrotome (LKB-2088) and stained with 1% toluidine blue (1% borax) on a 60°C hot plate for 2 min. Then ultra-thin slices were made and stained with uranyl acetate and lead citrate. The cells micro-structures were observed under TEM (JEM-1230, JEOL, Japan).
Enzyme-linked immunosorbent assay (ELISA) analysis
Mammary tissue samples were weighed and grinded with RIPA lysis buffer until homogenized. Then the homogenate was centrifuged at 12000rpm for 15min at 4°C, and the supernatant was collected in −20 °C until measurement. The expression of IL-1β were measured by ELISA and ac- cording to the manufacturer.
All data is shown as the mean± S.E.M, and experiments were independently repeated at least 3 times. One-way ANOVA and Dunnett's test were used for statistical analyses. Values of p<0.05 and were considered statistically significant.
High throughput sequencinganalysis
We first identified all statistically enriched terms (can be GO/KEGG terms, canonical pathways, hall mark gene sets, etc., based on what your choice during the analysis), accumulative hypergeometric p-values and enrichment factors were calculated and used for filtering. Remaining significant terms were then hierarchically clustered into a tree based on Kappa-statistical similarities among their gene memberships (similar to what is used in NCI DAVID site). Then 0.3 kappa score was applied as the threshold to cast the tree into term clusters. The terms within each cluster are exported in the Excel spreadsheet named “Enrichment Analysis”.
We then selected a subset of representative terms from this cluster and convert them into a network layout. More specifically, each term is represented by a circle node, where its size is proportional to the number of input genes fall into that term, and its color represent its cluster identity (i.e., nodes of the same color belong to the same cluster). Terms with a similarity score > 0.3 are linked by an edge (the thickness of the edge represents the similarity score). The network is visualized with Cytoscape (v3.1.2) with “force-directed” layout and with edge bundled for clarity. One term from each cluster is selected to have its term description shown as label.