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Fig. 8 | Cell Communication and Signaling

Fig. 8

From: Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

Fig. 8

Mutagenesis confirms the key residues. (a) MCF-7 cells were treated with LIP or other mutants. MCF-7 cells were plated in 96-well plates at a density of 5 × 104 cells/well and treated with 1 μg/mL LIP and mutants at 37 °C for 24 h. Cell death rates were analyzed by the LDH method. Each histogram represents the average value of triplicate experiments (**P < 0.01). Means ± SDs are shown. (b) Immunoblot analysis of LIP and mutants after incubation with MCF-7 cells. The observed bands represent the cell-bound protein from cell membranes. The band corresponding to polymer is indicated with black arrows. (c) Staining dead cells with propidium iodide (PI) for high content screening (magnification: 40×). MCF-7 cells were plated in 96-well plates at a density of 5 × 104 cells/well and treated with 1 μg/mL LIP and mutants for 24 h. Cells were washed twice with phosphate-buffered saline (PBS) and stained with PI and Hoechst (Sigma) for 20 min to visualize the cell nuclei. The samples were analyzed on a High Content Screen (PerkinElmer, USA). (d) Binding of Alexa488-labeled LIP and mutants to MCF-7 cells. (e) BIAcore diagrams of the binding of the D135A mutant of LIP to N003G. The D135A mutant abolished the binding to N003G

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