PMC

Schematic structure of Affitoxin.

A. 50 μg of purified Affitoxin was resolved in 4–12% Bis-Tris polyacrylamide gel along with pre-stained protein weight standards. B. MALDI-TOF MS of purified Affitoxin.

BT474 cells were treated with 2pM Affioxin in presence of 100nM Affibody and 100nM BSA. Cells were incubated for 72 hours and cell viability was measured using CellTiter-Glo. x

BT474 cells were treated with 43.5 pM Affitoxin for indicated time periods. After that medium containing Affitoxin was changed and incubation was continued up to 72 hours and cell viability was measured using CellTiter-Glo.

Binding of 0.32, 0.63, 1.25, 2.5, 5 nM wild type Z_HER2:342 Affibody molecule (A) and 6.17, 18.5, 55.5, 166.6, 500 nM of Affitoxin (B) to HER2/Fc on the sensor chip was tested using SPR-based binding assay. The red lines represent a global analysis of data using a Langmuir binding model. The Affibody molecule-HER2/Fc complex was allowed to dissociate for 2000 seconds in order to observe a measurable decayKinetic analysis of Z_HER2:342 Affibody molecule (A) or Affitoxin (B) binding to HER2/Fc-covered surface. Kinetic data are summarized in C.

A. Cells were incubated with 0.1 μM of Alexa Fluor® 488-labeled Affitoxin for 6 hours with and without Affibody molecules. B. HER2 expression level in five different breast cancer cell lines was determined by ELISA and is expressed in nanograms of HER2 per milligram of protein lysate. Cells were incubated with 5 μg/ml of Alexa Fluor® 488-labeled Affitoxin. Binding efficacy was determined by flow cytometry. C. BT474 cells were incubated with 5 μg/ml of Alexa Fluor® 488-labeled Affitoxin and increasing concentrations of Affibody. Binding efficacy was determined by flow cytometry and quantified by GraphPad software.

Five different breast cancer cells lines were transfected with pCMV-GLuc plasmid containing secreted Gaussia luciferase gene under control of strong CMV promoter. Gaussia luciferase activity was measured in the medium 24 hours after treatment with Affitoxin. The same cell lines were assessed for intracellular ATP level 72 hours following treatment (CellTiter-Glo, Promega).