Fig. 1

Silencing JAM-A re-sensitizes BT474 trastuzumab-resistant and SKBR3 lapatinib-resistant cells to anti-HER2 therapy. BT-474-trastuzumab-resistant cells (BT-474-Tr; A–C) or SK-BR-3-lapatinib-resistant cells (SK-BR-3-L; D–F) were plated in triplicate wells of 96-well plates (1500 cells per well) or in six-well plates (150,000 cells per well) and transfected the following day with 25 nM of control small interfering RNA (siRNA) (siNEG) or JAM-A siRNA; 24 h later, cells were treated with vehicle control (sterile nuclease-free water, 0.005% vol/vol or dimethyl sulfoxide, 0.0001% vol/vol), trastuzumab (100 μg/mL for BT-474-Tr cells), or lapatinib (500 nM for SK-BR-3-L cells); 72 h later, cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay or cells were harvested for Western blot analysis. (a) Cell viability response of BT-474-Tr cells to trastuzumab treatment alone and combined with JAM-A knockdown. (b) Representative Western blot images for expression of HER2, pAkt, and pERK following specified treatments in BT-474-Tr cells. (c) Densitometry analysis of HER2, pAKT, and pERK expression in BT-474-Tr cells following specified treatments. Silencing JAM-A in addition to trastuzumab treatment reduced HER2 expression and Akt phosphorylation more than trastuzumab treatment alone in BT474-Tr cells. (d) Cell viability response of SK-BR-3-L cells to lapatinib treatment alone and combined with JAM-A knockdown. Silencing JAM-A expression in addition to anti-HER2 treatment was more effective at reducing resistant cell viability than anti-HER2 treatment alone. (e) Representative Western blot images for expression of HER2, pAkt, and pERK following specified treatments in SK-BR-3-L cells. (f) Densitometry analysis of HER2, pAKT, and pERK expression in SK-BR-3-L cells following specified treatments. Silencing JAM-A in addition to lapatinib treatment reduced Akt phosphorylation more than lapatinib treatment alone in SK-BR-3-L cells (*P <0.05, **P <0.01, ***P <0.001 by one-way analysis of variance with Tukey’s multiple comparison test, n = 3 independent experiments).