Fig. 6: Effect of DpPorA DE peptide on MDA-MB-231 cells.

a Cell viability was assessed by MTT assay 24 h post-treatment with 10 µM, 20 µM, and 25 µM DpPorA DE peptide. Data represents mean ± SEM from n = 4 (0 µM, 10 µM and 20 µM) and n = 3 (25 µM). Each dot represents a biological replicate, where each replicate was an independently seeded and treated culture of MDA-MB-231 cells on different days with different passage numbers. Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test comparing each treatment to the untreated control (0 µM); Adjusted p values correspond to *p = 0.0206 (10 µM), ***p = 0.0006 (20 µM), ****p < 0.0001 (25 µM) vs. 0 µM. b Immunofluorescence staining with CellMask Deep Red was used to evaluate the integrity of the cell membrane in Control, 0.00125% DDM and 25 µM DpPorA DE-treated cells. Cells were categorized into two phenotypes based on membrane integrity: Intact and Disrupted. Control cells displayed a mixture of phenotypes with a distribution of 67.38 % Intact and 32.62 % Disrupted. c DpPorA DE-treated cells showed a significant increase in the Disrupted phenotype, with 99.3% of cells being Disrupted, indicating a substantial impact of the DpPorA DE peptide on cell membrane integrity. d Fluorescence microscopy images showing different cell membrane integrity phenotypes in Control, 0.00125% DDM, and 25 µM DpPorA DE peptide-treated cells. The two different phenotypes are represented as a hashtag for Intact and the arrow indicates Disrupted cells (100 cells per group, scale bar 20 μm, magnification 63×). e Representative fluorescence images showing the distribution of 5-FAM-DpPorA DE (green) in MDA-MB-231 cells at 4 h and 24 h post-addition of peptide. Cell membranes were stained with CellMask (red) (50 cells per group, scale bar 20 μm, magnification 63×). f Single-cell fluorescence intensity scatter plot showing increased incorporation of 5-FAM-DpPorA DE at 24 h compared to 4 h, indicating time-dependent peptide incorporation in the cell population.