Fig 1.
Histatin-1 improves rates of wound closure in an in vitro scratch assay.
(A) Representative images from wound healing assay of HCLE cell cultures treated with histatin-1 demonstrating enhancement of wound closure compared to “vehicle only” (PBS) control; scale bar = 200μM (only 10 μM shown) (B) Summary bar graph illustrating percentage wound closure at indicated time points during the scratch assay. Notable is the statistically significant improvement in wound closure times. Each condition was compared to “vehicle only” (PBS) control. P values: ***P ≤ 0.001 as determined by two-way ANOVA with Bonferroni’s posttest. The error bar represents means ± SEM of three scratched areas imaged per condition.
Fig 2.
Histatin-1 enhances pathfinding of HCLE cells across a scratch wound in vitro.
(A) Migratory paths of individual cells along the edge of the wound, showing shorter path length with histatin-1 vs. “vehicle only” (PBS) control; scale bar = 200μM (only 10 μM shown) (B) Histatin-1 reduces path length to wound closure in comparison to “vehicle only” (PBS) control. Each condition was compared to “vehicle only” (PBS) control. Length of the path from one side of the wound was divided by half the linear width of the wound in order to give a ratio that represents the non-random/efficiency of epithelial migration across the cell free area of the scratch. P values: ** P ≤ 0.01using one-way ANOVA with Bonferrroni’s multiple comparison test. The error bar represents means ± SEM of three scratched areas imaged per condition.
Fig 3.
Histatin-1 enhances cell spreading.
Cell spreading assay using cell surface area measurements and visualization of actin in HCLE cells via phalloidin staining. Noted is the statistically significant increase in cell surface area after application of histatin-1 compared with “vehicle only” (PBS) control. (A) Representative images of HCLE cell spreading 24 hours after seeding in the presence or absence (“vehicle only” (PBS) Control) of histatin-1. Green, actin; blue, nuclei. Scale bar = 20μM. (only 10 μM shown) (B) Average surface area per cell was quantified from images similar to those in A. Measurement of average individual cell surface area is significantly higher with all tested concentrations of histatin-1 treatment. Each condition was compared to “vehicle only” (PBS) control. P values: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.0001 using student’s T test. The error bar represents means ± SEM; (n = 60 “vehicle only” (PBS) control n = 60 for 5μM, n = 62 for 10 μM, n = 60 for 50 μM of histatin-1 treatment).
Fig 4.
MTT assay for cell metabolic activity.
Noted is significant increase in cell metabolic activity with histatin-1 application compared with “vehicle only” (PBS) control upto concentrations of 200 μM, and a significant decrease in metabolic activity at 400 μM. Each condition was compared to “vehicle only” (PBS) control. P values: ** P ≤ 0.01, *** P ≤ 0.0001 using one-way ANOVA with Bonferrroni’s multiple comparison test. The error bar represents means ± SEM of the triplicate measurement.
Fig 5.
BrdU assay for cell proliferation.
Noted is no statistically significant increase in cell proliferation in the BrdU incorporation assay at any tested concentraton. Each condition was compared to “vehicle only” (PBS) control. The error bar represents means ± SEM of the triplicate measurement.
Fig 6.
Demonstrates a lack of significant toxicity to HCLE cells after application of histatin-1 compared with “vehicle only” (PBS) control. Each condition was compared to “vehicle only” (PBS) control. There were no statistically significant differences noted between “vehicle only” (PBS) control and the histatin peptides. The error bar represents means ± SEM of the triplicate measurement. 100% lysis refers to the manufacture’s supplied lysis buffer, causing 100% cell death.