%0 Journal Article
%T Neutrophil TLR4 and PKR are targets of breast cancer cell glycosaminoglycans and effectors of glycosaminoglycan-induced APRIL secretion
%A Brianna-Marie Lee
%A Eric Quan
%A Jesusa L. Rosales
%A Kelvin Chan
%A Ki-Young Lee
%A Uilst Bat-Erdene
%A Wejdan Matook
%J Archive of "Oncogenesis".
%D 2018
%R 10.1038/s41389-018-0058-2
%X Following approval by the University of Calgary¡¯s Conjoint Health Research Ethics Board, blood was obtained from healthy volunteers that gave informed consent. Neutrophils were isolated as described previously48,49 and suspended in Hank¡¯s Balanced Salt Solution with Ca2+/Mg2+ (Gibco). BCCs (Hs578T, SKBR3, T47D, and MCF7) obtained from the American Type Culture Collection (not authenticated but tested for mycoplasma in the current year) were cultured in Dulbecco¡¯s Modified Eagle Medium (Gibco) with 10% fetal bovine serum (Gibco) and 100£¿U/ml each of penicillin and streptomycin (Gibco) in 5% CO2 at 37£¿¡ãC. a APRIL stalk is clearly detectable in neutrophils but not in breast cancer cells. On the left panel, lysates of MCF7, T47D, Hs578T, and SKBR3 cells (60£¿¦Ìg each), as well as neutrophils (5£¿¦Ìg) were subjected to SDS-PAGE and western blotting using APRIL stalk antibody (ED, MyBioSource: MBS194153). On the right panel, neutrophils and T47D cells [non-transfected and transfected with pCR3 (T47D-pCR3) or pCR3-soluble APRIL (T47D-pCR3-sAPRIL)] were immunoblotted using Aprily-5 (Novus Biologicals: NBP1-97599; top panel) and reblotted using April ED (middle panel) antibodies. Aprily-5 detects soluble APRIL. The bottom panel shows Ponceau S staining of the membranes. b Breast cancer cells enhance APRIL secretion from neutrophils. Neutrophils (2£¿¡Á£¿106) and breast cancer cells (5£¿¡Á£¿105) were incubated alone or together. Cells were then pelleted and supernatants were analyzed for secreted neutrophil APRIL by direct ELISA. Recombinant APRIL (Alexis Corp.) was used as standard. Anti-APRIL (H-60, Santa Cruz Biotech.:sc-28919) used as detection antibody was complexed with HRP-linked anti-rabbit IgG (Cell Signaling). Immunoreactivity was detected using 3,3¡ä,5,5¡ä-tetramethylbenzidine liquid substrate (Sigma). The colorimetric reaction was stopped using 2£¿M HCl and measured at 490£¿nm using a SpectraMax M2/M2e multi-mode microplate reader. Measured APRIL in 1£¿ml corresponds to secretion from 1£¿¡Á£¿106 neutrophils. Note that measured levels of APRIL from 5£¿¡Á£¿105 and 2£¿¡Á£¿106 breast cancer cells were similar and reflect background values. c Breast cancer cell-associated and -released factors are involved in stimulating neutrophil APRIL secretion. Neutrophils (2£¿¡Á£¿106) and T47D BCCs (5£¿¡Á£¿105) were co-incubated in the presence or absence of transwell membranes (3£¿¦Ìm pore size; Corning) in 24-well plates. When transwell membranes were used, BCCs were loaded in the upper chamber and neutrophils in the lower chamber. Following incubation for 5£¿min at 37£¿¡ãC, cells were pelleted and
%U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002394/