Stemness of PN-transfected BCA cells was determined by staining with anti-CD24 and anti-CD44 antibodies and analyzed by flow cytometry and compared with mock-transfected cells in conditions without or with anti-PN peptide. Briefly, BCA cells were seeded at the concentration 1 × 105 cells into 6-well plates with 2 ml of complete media. Cell media was removed the next day and changed to 1% FBS media for 24 h. Then, the treatment in 1% FBS media without or with anti-PN peptide (1 μM) were refilled and incubated for a further 24 h. At the end of treatment, cell pellets were collected and incubated in 2% FBS/1x PBS with 1:20 dilution of FITC-labeled anti-CD24 antibody (cat no. 21270043, ImmunoTools GmbH, Friesoythe, Germany) and 1:5 dilution of allophycocyanin (APC)-labeled anti-CD44 antibody (cat no. 21270446, ImmunoTools) for 30 min. CytoFLEX® Flow cytometry (Beckman Coulter, Inc. Brea, CA, USA) and CytExpert® software version 2.1 (Beckman Coulter, Inc.) were used for analysis.

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