Cells are seeded in a 96-well plate at a density of 2,000 cells per well and grown overnight at 37°C, 5% CO₂ in the media described for each cell line above. Cells are subsequently treated with 100 μL of fresh media, containing varying concentrations of knottin-Fc fusion proteins or linker-modified MMAF, and incubated for 5 days at 37°C, 5% CO₂. Cell proliferation is measured using the Cell Counting Kit-8 (CCK-8), by adding the water-soluble tetrazolium salt, WST-8, to each well in an amount equal to 10% of the culture volume. After incubation for 1 hour at 37°C, absorbance at 450 nm is measured with a Synergy H4 microtiter plate reader. Cell proliferation is expressed as a percentage of absorbance relative to the control of untreated cells. Percent maximum proliferation is then reported as (sample − background)/(control − background) × 100. Error bars represent the SD of experiments performed in triplicate.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Currier NV, et al. Targeted Drug Delivery with an Integrin-Binding Knottin-Fc-MMAF Conjugate Produced by Cell-Free Protein Synthesis. Mol Cancer Ther. 2016 Jun;15(6):1291-300.

745.99

C₄₀H₆₇N₅O₈

863971-12-4

CC[C@H](C)[C@@H]([C@@H](CC(N1[C@@H](CCC1)[C@@H]([C@@H](C)C(N[C@H](C(OC)=O)CC2=CC=CC=C2)=O)OC)=O)OC)N(C([C@H](C(C)C)NC([C@H](C(C)C)NC)=O)=O)C

Room temperature in continental US; may vary elsewhere