Cell cultures of BCA cells

BCA cell lines, MDA-MB-231 and MCF-7 were used in the study. They were cultured with Dulbecco Modified Eagle’s Medium (DMEM) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). These media contained 10% fetal bovine serum (FBS) (Gibco), using penicillin/streptomycin (Gibco) as antibiotics and amphotericin B (Gibco) as an antifungal drug with 5% CO₂ and 90% humidity at 37 °C.

Lipofectamine™ 3000 (Invitrogen, Thermo Fisher Scientific) was used to transfect the blank pCDNA™3.1 plasmid (v385–20, Invitrogen) or pCDNA™3.1 PN-plasmid into BCA cell lines. After transfection, the cells were selected by Geneticin™ (Gibco) (up to 1 mg/ml) to create stable cell lines. PN and integrin expressions were tested by reverse transcriptase (RT)-polymerase chain reaction (PCR) using Light Cycler® 480 II system (Roche, Basel, Switzerland) with specific primers (Table 1) [9, 32]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression was used as an internal control (Table S1). The cycle threshold (Ct) value was used for calculation of expression folding.

Correlation between tissue PN expression and serum PN level in BCA patients (P-value = 0.010)