A study in Cancer Cell International discusses the role of upregulation of NF-kappa-β-Inducing Kinase (NIK) protein in a high malignancy breast cancer cell line. NIK has the potential to be a diagnostic marker of breast cancer, suggest the authors.
Breast cancer cells were plated on the 12-well plates and incubated overnight. After incubation, each cell was washed once with D-PBS and incubated for 5 minutes in 0.25% Trypsin-EDTA . Then the normal culture medium of RPMI-1640 was added to each well up to 1 mL. The cell number of each sample was calculated by loading the cell suspension medium into a hemocytometer.RPMI culture medium containing 0.3% agarose with LM05-shGFP or LM05-shNIK cells over a bottom layer of 0.
For immunohistochemistry analysis, antigen retrieval was performed in 10 mM citrate buffer at 120 °C or 95 °C for 20 min. After cooling to room temperature, the endogenous peroxidase activity was blocked with 3% H for 30 min. Then, these sections were incubated in 2.5% normal horse serum for 1 h at room temperature. The specimens were incubated with primary antibodies against α-SMA , Ki-67 , CAM5.2 , NIK and cIAP1 at 4 °C overnight.
For hematoxylin and eosin staining, deparaffinized and rehydrated sections were stained in Mayer's hematoxylin solution for 10 min. Then, the sections were soaked in 0.1% saturated lithium carbonate at 37 °C for 5 min. After washing in distilled HO, the sections were stained with eosin solution for 10 min. Then, the sections were immediately washed with 100% ethanol, 90% ethanol and xylene. Finally, the sections were mounted, and images were acquired with a BZ-X700 microscope.
For terminal deoxynucleotidyl transferase dUTP nick end labeling staining, deparaffinized and rehydrated sections were stained with a TUNEL Assay Kit according to the manufacturer’s protocols. After staining with hematoxylin, dehydration and mounting, images were acquired with a BZ-X700 microscope.Total RNA was extracted with QIAzol according to the manufacturer’s protocol.
The raw sequence data were trimmed to remove adapter sequences by Trim-Galore , and then, the trimmed read data were mapped on GRCh38/hg38 using HISAT2 . After the SAM files were converted to Bam files using SAMtool , the gene expression FPKM was quantified by StringTie . Then, read count matrices were generated for each gene by prepDE.py of StringTie. After removing low-expression genes with less than 10 counts per million , differentially expressed genes were identified using edgeR in R.
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