Fig. 3

JAM-A cleavage is augmented in BT-474 trastuzumab-resistant and SK-BR-3 lapatinib-resistant cells compared with their corresponding drug-sensitive cell lines. BT-474 trastuzumab-sensitive (BT-474-Sens), -resistant (BT-474-Tr), SK-BR-3 lapatinib-sensitive (SK-BR-3-Sens), and -resistant (SK-BR-3-L) cells were plated at 150,000 cells per well in six-well plates for Western blot analysis and at 50,000 cells per well in 24-well plates for quantitative polymerase chain reaction (qPCR) analysis; 48 h later, cells designated for cleaved JAM-A (cJAM-A) detection were washed and serum-free media was added to the wells. At 72 h, conditioned media was centrifuged to exclude any floating cells and concentrated using Amicon Ultra-2 Centrifugal Filter Spin Columns, whereupon cells were harvested for Western blot and qPCR analysis. (a) qPCR analysis of JAM-A mRNA expression in BT-474-Sens and BT-474-Tr cells. (b) Representative Western blot images and densitometric analysis of basal JAM-A protein expression in BT-474-Sens and BT-474-Tr cells (relative to actin). (c) Representative Western blot images and densitometric analysis of basal cleaved JAM-A released from BT-474-Sens and BT-474-Tr cells (relative to Ponceau S). (d) qPCR analysis of JAM-A mRNA expression in SK-BR-3-Sens and SK-BR-3-L cells. (e) Representative Western blot images and densitometric analysis of basal JAM-A protein expression in SK-BR-3-Sens and SK-BR-3-L cells (relative to actin). (f) Representative Western blot images and densitometric analysis of basal cleaved JAM-A released from B SK-BR-3-Sens and SK-BR-3-L cells (relative to Ponceau S). JAM-A cleavage and release of cleaved JAM-A was significantly augmented in BT-474-Tr and SK-BR-3-L cells compared with their corresponding drug-sensitive cell lines. *P <0.05 by equal variance unpaired t test, n = 3 independent experiments