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. 2016 Mar 6;7(14):18851–18864. doi: 10.18632/oncotarget.7944

Figure 3. BAG-1 overexpression attenuates the growth inhibitory effect of trastuzumab while BAG-1 knockdown potentiates the effect of trastuzumab.

Figure 3

(A) A representative immunoblot shows BAG-1 and HER2 protein expression in SKBR3 cells 96 h post-transfection with the indicated constructs; β-actin was used as a loading control. Bar graph shows the effect of trastuzumab (8 μg/ml) on the long-term growth potential of SKBR3 cells transfected with vector control (pcDNA3) or pcDNA3 containing BAG-1S or BAG-1S H3AB (BAG domain mutant incapable of interacting with HSC70). Transfectants were maintained in complete media supplemented with G418 for 2 weeks. Data on the bar graph show the mean ± SD and are representative of two independent experiments, each with three technical replicates. Unpaired t-test was used for comparison between different treatments; **p < 0.01, ***p < 0.001. (B) A representative immunoblot shows BAG-1 and HER2 expression levels in SKBR3 and BT474 cells 3 days following siRNA (50 nM) knockdown; β-actin was used as a loading control. Densitometric analysis of immunoblots from 3 independent experiments was used to determine expression of total BAG-1 protein and is expressed as a percentage of the maximal value. The amount of BAG-1 protein knockdown by siBAG-1 is expressed as a percentage of the corresponding NSC treatment for each cell line. (C) SKBR3 and BT474 cells transfected with NSC or siBAG-1 were grown for 5 days with or without trastuzumab (32 μg/ml) and cell growth was measured using crystal violet assay. Data are the mean ± SEM from 4 independent experiments, each with three technical replicates. Unpaired t-test was used for comparison between different treatments; *p < 0.05, **p < 0.01, ***p < 0.001. (D) A representative immunoblot shows induction of Tet-On myc-BAG-1S using an anti-myc antibody in SKBR3 cell clones 10 and 11 48 h after addition of doxycycline (2 μg/ml); β-actin was used as a loading control. (E) Line graph shows the effect of BAG-1S induction by doxycycline (2 μg/ml) on cell growth over 9 days using crystal violet assay. Data shown on the graph are the mean ± SEM from 3 independent experiments, each with three technical replicates. Unpaired t-test was used for comparison between doxycycline induced and uninduced cells; ***p < 0.001.