Fig 1: Cell pattern diagram of B4GALNT2 interacting with HLA-B protein to promote breast cancer cell proliferation, migration and invasion.
Fig 2: Construction of the cell model. (A) Lentiviral transfection efficiency of two breast cancer cell lines. (B, C) The mRNA and protein expression levels of B4GALNT2 gene were detected by RT-qPCR and Western blot after 72h of transfection with lentivirus. The success of the model construction is reflected in the joint results of (B, C). ***p < 0.001, ****p < 0.0001.
Fig 3: The effects of B4GALNT2 gene on the cell proliferation, invasion, migration and cycle of breast cancer cell lines were investigated by in-vitro cell experiments. (A) Using Celigo instrument, through counting for 5 consecutive days, it was found that the proliferation ability of the cells of the shB4GALNT2 group were significantly lower than that of the shCtrl group; (B) MTT assay was used for detection of the cell viability. After 5 consecutive days of detection, it was observed that the cell viability of shB4GALNT2 group was significantly lower than that of shCtrl group. (C) Flow cytometry was used for determining the early apoptosis rate of cells transfected with lentivirus, where it was found that the apoptosis rate of cells in shB4GALNT2 group was higher. (D, E) Transwell assay was used for measuring the migration and invasion abilities of cells after knockdown of B4GALNT2 gene, where it was found that the migration and invasion ability of cells in shB4GALNT2 group were significantly reduced. (F, G) Flow cytometry was used for detecting the cell cycle, where it was found that the B4GALNT2 gene knockdown resulted in significant G1 phase arrest. **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig 4: B4GALNT2 gene is highly expressed in breast cancer tissues and cell lines. (A) The volcano map of the differential expression of genes between 1094 breast cancer tissues and non-tumor tissues (red corresponds to p<0.05; black corresponds to p>0.05). (B) A paired diagram for the expression levels of the B4GALNT2 gene in 106 pairs of breast cancer and normal adjacent tissues. (C) The relative expression level of gene B4GALNT2 in five different breast cancer cell lines. *p < 0.05.
Fig 5: The MDA-MB-231 cell line was used to verify the functional restoration of HLA-B interaction protein genes. (A) The proliferation ability of the cells was evaluated with Celigo apparatus for 5 consecutive days. (B, C) Transwell plate and MTT reagent were used to detect cell migration and cell viability, respectively. These experiments showed that HLA-B protein could restore the proliferation and migration abilities of breast cancer cells, thus further verifying the existence of an interaction between B4GALNT2 and HLA-B protein molecules. (A) Scale bar=100 µm; (B) Scale bar=50 µm. Note: NC+NC=normal control group; KD+OE=knockdown of the B4GALNT2 gene + the overexpressed HLA-B gene; KD+NC=knockdown of the B4GALNT2 gene, only. Note: for the 4 positive interaction proteins identified (CLU, AXL, HLA-B and EIF4A2), an interference or expression vector was constructed, thus achieving overexpression of the positive interaction protein while inducing interference with the target gene in a cell line. The Celigo instrument scanned the changes in cell proliferation ability for 5 consecutive days, and verified the functional restoration of the downstream mechanism genes on the target gene. Only the HLA-B gene had an obvious functional restoration effect. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Supplier Page from MilliporeSigma for Anti-B4GALNT2 antibody produced in rabbit