Fig 1: CRISPR-induced downregulation of pLXNB3 negatively impacts TNBC growth in vitro and in vivo. (A) CRISPR KD (polyclonal population) in MDA-MB468 cells results in reduced expression of the PLXNB3 protein compared to control conditions as determined by western blotting (sg1 and sg2: guide RNAs targeting PLXNB3; GFP: cells transfected with a guide RNA against GFP; Cas9: cells transfected with a vector only containing Cas9; NT: nontreated cells). (B) PLXNB3 KD using CRISPR-Cas9 negatively impacts 2D cancer cell growth as determined by the growth curve assay compared to sgGFP, Cas9, and NT controls. Average values with standard deviation are represented (n = 6). P-values from Tukey’s multiple comparisons test against sgGFP control are reported. (C) PLXNB3 KD using CRISPR-Cas9 negatively impacts the in vivo tumor growth ability of MDA-MB468 cells (polyclonal population) grown subcutaneously in NSG mice. Left to right: Tumor volume (average values with standard deviation, n = 5), tumor weight, and picture of tumors at sacrifice. P-values from Tukey’s multiple comparison test against sgGFP control are reported. See also Figure S4.
Fig 2: siRNA-mediated knockdown of PLXNB3 negatively impacts TNBC growth and invasion in vitro. (A) PLXNB3 KD using 5 nM siRNA negatively impacts the 2D cell growth of PLXNB3-positive cells, as determined using the growth curve assay. The PLXNB3-negative TNBC cell line HCC1937 and the control cell line MCF10A were used as negative controls. Average values with standard deviation are represented (n = 3). P-values from Tukey’s multiple comparisons test against Scr control are reported. NT = nontreated cells. (B) PLXNB3 KD (5 nM siRNA) negatively impacts TNBC cell ability to undergo unlimited cell divisions as determined using the colony forming assay. The PLXNB3-negative cell line (HCC1937) was used as a negative control. Scale bar = 5 mm. (C) PLXNB3 KD (5 nM siRNA) negatively impacts TNBC cell ability to invade Matrigel as determined using the 3D spheroid growth assay (n = 6). The number of invading cells was determined using ImageJ. P-values from Tukey’s multiple comparisons test against Scr control are reported. See also Figure S3.
Fig 3: Identification of PLXNB3 as a novel TNBC-associated surface protein. (A) Data mining and functional characterization workflow. The list of TNBC-enriched N-glycoproteins was restricted to include only cell surface proteins31 and was subsequently ranked based on the lack of detection in normal tissue according to the Human Protein Atlas.32 PLXNB3 was the top-ranking candidate that had not previously been investigated in the context of breast cancer and hence was selected for functional interrogation. (B) Volcano plot highlighting differentially expressed TNBC and NC proteins. Cutoff values (red dotted lines): log2 fold change > 2 (vertical) and adjusted p-value < 0.05 (horizontal). The color of dots represents the number of TNBC cell lines positive for a respective protein and minus the number of positive NC cell lines. Intense red signifies proteins detected exclusively in TNBC samples, whereas intense blue signifies proteins exclusively detected in NC cells. (C) TNBC-enriched cell surface proteins ranked based on the lack of immunohistochemistry (IHC) detection in normal tissue. The proportion of normal tissue with high, medium, low, not detected, and NA IHC staining annotations was calculated from the Human Protein Atlas (version 20.1) data. (D) PLXNB3 expression levels in the analyzed cell lines as determined using the glycoproteomics method. N.D. = not detected. (E) Overall survival of breast cancer patients (n = 1079) based on PLXNB3 mRNA expression levels.35 Median PLXNB3 mRNA expression levels were used as the cutoff for high and low expressions. See also Figure S2.
Supplier Page from OriGene Technologies for PLXNB3 Human siRNA Oligo Duplex (Locus ID 5365)