Fig. 3.

Notch-1-mediated inhibition of PTEN is necessary for trastuzumab-resistant bulk cell proliferation a–d. BT474TR or HCC1954 cells were transfected with control siRNA (SCBi), Notch-1 siRNA, and/or PTEN siRNA for 48 h. Post transfection, cells were seeded at 100,000 cells/well in a 6 well plate and treated daily with PBS (a, c) or 20 μg/mL trastuzumab (b, d) for 10 days. Post treatment, cells were collected and counted. Mean fold increase in live cells was calculated as the number of live cells harvested at day 10/number of live cells seeded at day 0. Asterisk (*) denotes statistical significance compared to SCBi or (**) Notch-1i as calculated using a one-way ANOVA with an overall statistical significance of p < 0.0001 from three independent experiments. e Total cellular Notch-1, PTEN, Akt, β-Actin, phosphorylation of Akt-1 at threonine308 (PThr308-Akt1) and serine473 (PSer473-Akt1) protein expression from the BT474 TR lysates in a, b post 48 h PBS (left) or trastuzumab (right) treatment was detected by Western blot. Mean values from densitometric analysis of Notch-1, PTEN, and PSer473-Akt1 normalized to β-Actin or Akt, respectively, are displayed below their respective blots, and quantitated from three independent experiments using Image J