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. Author manuscript; available in PMC: 2014 Aug 15.
Published in final edited form as: Nat Commun. 2014 Jul 22;5:4478. doi: 10.1038/ncomms5478

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

Figure 5

(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 medium containing each peptide at the indicated concentrations at 37°C for 6 hours. ATP levels were measured by CellTiter Glo (Promega). DMSO alone at 37 °C for 6 hours 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 hours. (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) and HLA-z13 (25 μg/ml) (red) at 37 °C for 6 hours. 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 medium containing z13 peptide with or without an apoptosis-inducing peptide and/or endosome-escaping peptide. Cells were cultured at 37°C for 20 hours in medium containing: no peptide (a); z13, 100 μg/ml (b); dKLAK, 100 μg/ml (c); dKLAK-z13, 100 μg/ml (d); HLAH-z13, 25 μg/ml (e); dKLAK-z13, 100 μg/ml, plus HLAH-z13, 25 μg/ml (f). Scale bars in all subpanels 200 μm. (e) TUNEL assay of CNGB3-Myc transfected HEK293T cells treated with a mixture of dKLAK-z13 (100 μg/ml) and HLAH-z13 (25 μg/ml) for the periods indicated. Scale bars in all subpanels 100 μm.