Table 1.
Name and corresponding treatment of BT474 derivatives, all with 30 weeks of treatment.
Fig 1.
Phenotype of long-term siRNA treated cells.
When seeded at the same density for 5 days without treatment, BT474-TNP-H30 grew slower than parental BT474 and other derivatives. Images showing representative fields at 100X magnification (A). Fold change in nuclei count after 5 days of growth (B) and the corresponding doubling time (C). Bars represent mean ± SD of 6 replicates in a 96-well plate. Asterisks * indicate statistical significance when compared to the parental BT474 (P < 0.05).
Fig 2.
EMT and TIC characteristics of long-term HER2 siRNA treated cells.
Western blot showed no concurrent downregulation of E-cadherin and upregulation of vimentin in BT474 derivatives compared to parental BT474, indicating no evidence of epithelial to mesenchymal transition (A). Flow cytometry was used to determine the surface expression of CD24 and CD44 among the BT474 derivatives. There was no enrichment of tumor initiating cells (CD24-/CD44+) in the long-term treated cells (B).
Fig 3.
Cell viability of BT474 derivatives when challenged with their respective long-term treatment regimens.
Long-term siHER2 treated BT474 derivatives were challenged with HER2 siRNA delivered using either DharmaFECT (A), nanoparticles (NP) (B), or trastuzumab conjugated nanoparticles (TNP) (C). Their response to trastuzumab (D–F) or lapatinib (G–I) was compared to those of the resistant derivatives BT474-TR and BT474-LR.
Fig 4.
Cell viability of BT474 derivatives at earlier time points during repeated HER2 siRNA treatment.
BT474 derivatives were challenged with HER2 siRNA delivered using nanoparticles (A and C), or trastuzumab conjugated nanoparticles (B and D) after 8 weeks (A and B) or 15 weeks (C and D) of treatment.
Fig 5.
Heat map of selected genes that were differentially expressed as determined by RPPA.
Proteins from BT474 derivatives while under their corresponding long-term treatment agents were spotted onto reverse phase protein microarrays and probed using a panel of antibodies. Data are presented in triplicate, with each data point representing the expression of a given protein from a single replicate. Color gradient represents protein expression levels (log base 2). The original full panel raw data are available as S1 Dataset.