Figure 5. Z13-targeted apoptosis of CNGB3-expressing cells in vitro.

(a) Cell viability assays of CNGB3-positive A431 cells (left) and control CNGB3-negative A431 cells (right) after incubating cells with z13, dKLAK-z13, HLAH-z13 or dKLAK-z13 plus HLAH-z13. Cells were cultured in a medium containing each peptide at the indicated concentrations at 37 °C for 6 h. ATP levels were measured by CellTiter Glo (Promega). dimethylsulphoxide alone at 37 °C for 6 h at a final concentration of 0.01% had no effect by this assay (data not shown). (b) Number of living CNGB3-positive and -negative A431 cells following treatment with the indicated reagents for 20 h. (c) Inhibition of the activity by KLA-z13 plus HLA-z13 by palmitoyl-z13 peptide. CNGB3-expressing A431 cells were treated with C16-z13 alone (blue) or with a mixture of KLAK-z13 (100 μg ml⁻¹) and HLA-z13 (25 μg ml⁻¹; red) at 37 °C for 6 h. Each data point represents average of triplicate measurements. This analysis was repeated three times. (d) TUNEL assay of CNGB3–MYC-expressing HEK293T cells cultured in a medium containing z13 peptide with or without an apoptosis-inducing peptide and/or endosome-escaping peptide. Cells were cultured at 37 °C for 20 h in a medium containing: no peptide (a); z13, 100 μg ml⁻¹ (b); dKLAK, 100 μg ml⁻¹ (c); dKLAK-z13, 100 μg ml⁻¹ (d); HLAH-z13, 25 μg ml⁻¹ (e); and dKLAK-z13, 100 μg ml⁻¹, plus HLAH-z13, 25 μg ml⁻¹ (f). Scale bar, 100 μm. (e) TUNEL assay of CNGB3–MYC-transfected HEK293T cells treated with a mixture of dKLAK-z13 (100 μg ml⁻¹) and HLAH-z13 (25 μg ml⁻¹) for the periods indicated. Scale bar, 100 μm.