Fig 1.
i. Structures of antifungal β-peptides used in this study.
The codification (#number) corresponds to the nomenclature used by Lee et al. in a previous publication [11]. β-peptide (e) is a fluorescently labeled β-peptide. The C-terminus is amidated for all β-peptides. ii. HPLC-RP retention time for the β-peptides synthesized in this work, see Figs A-D in S1 File for further details.
Fig 2.
Panel A and B represent the curves for HepG2 (▪) and Caco-2 (o), exposed to the least and most hydrophobic β-peptide (#4 and #16, respectively) during 24 hours respectively. Panels C and D feature the behavior of Caco-2 and HepG2 correspondingly, for the three ACHC-containing β-peptides. The curves represent the mean of two independent experiments, each with four biological replicates. The error bars correspond to the standard error of the mean, where n = 8.
Fig 3.
PCA of β-peptide concentration and viability of cells by each different hydrophobic β-peptides.
Panels a and b are for HepG2 and Caco-2, respectively.
Table 1.
β-peptides selected for the study and analysis of their cytotoxicity towards HepG2 and Caco-2 cells compared to their antifungal activities.
Fig 4.
Confocal images for Caco-2 cells.
(a-d) untreated cells, (e-h) cells treated with 16 μg/mL of (ACHC-β3hV-β3hK), and (i-l) cells treated with 16 μg/mL of coumarin-(ACHC-β3hV-β3hK). (a, e, i) show phase-contrast images, (b, f, j) show the signal emission for the 545 (green) and 405 (blue) nm filters, (c, g, k) show signals for the 640 (red) and 408 (blue) nm filters, and (d, h, l) show all signals. These are representative images of at least three independent experiments. The exposure time was 1 hour. Cells were stained for 30 min as described in the methodology.
Fig 5.
(a-d) untreated cells, (e-h) cells treated with Triton X-100 at 0.05% v/v, (i-l) cells treated with 16 μg/mL of (ACHC-β3hV-β3hK), and (m-p) cells treated with 16 μg/mL of coumarin-(ACHC-β3hV-β3hK). (a, e, i, m) show phase-contrast images, (b, f, j, n) show signal emissions for the 545 (green) and 405 (blue) nm filters, (c, g, k, o) show signals for the 640 (red) and 408 (blue) nm filters, and (d, h, l, p) show all signals. The oval encircles a zone where the β-peptide was detected, and co-localized with cell membrane, and the arrow points to the nucleus of the cell affected by the β-peptide.
Fig 6.
HepG2 exposure for 1 hour to 16 μg/mL of coumarin-(ACHC-β3hV-β3hK).
The images were captured increasing Z to 0.5 μm from left to the right per plane. The experiments were conducted at least two independent times. The blue represents coumarin signal at 408 nm captured. Panels (e-h & m-p) show coumarin (blue) signals co-localization with structures stained with the cell membrane dye (red).
Fig 7.
Acridine orange (AO) uptake in HepG2 and Caco-2.
The (a, c, e & g represents untreated cells, and (b, d, f and h) represents cells treated with 16 μg/mL of (ACHC-β3hV-β3hK) for 3 hours in suspension under incubation conditions. FL1-H represents nucleic acids stained with AO while FL3-H represents lysosomal uptake of AO. The experiment was performed at least two independent times and the measurement has two biological replicate the figure presented is a representative result obtained from the assays performed.