Fig. 5

Flow cytometric analysis on the apoptotic effect of RALA/ALN NPs in a LN229 and b T98G cells. RALA/ALN NPs prepared at mass ratio 10:1, to achieve a final ALN concentration of 43.83 μM and 60.55 μM in T98G and LN229 cell lines, respectively. Cells were treated for 6 h before medium was removed and replaced with complete fresh media. Cells were left to incubate for 72 h prior to analysis. Cells were prepared for flow cytometry using the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences Pharmingen™, USA) and PI staining as per manufacturers’ guidelines and analysed using the Accuri C6 Plus (BD Bioscience, UK) flow cytometer. Results are reported as mean ± SEM, n=3. Confocal microscopy images of H-Ras plasma membrane localisation in c LN229 and d T98G cells. Cells were transfected with 0.5 μg/μL plasmid EGFP-H-Ras for 4 h using the RALA peptide delivery system at N:P 10. After 24 h, cells were treated with either ALN or RALA/ALN NPs at a mass ratio of 10:1 (conc. 43.83 μM and 60.55 μM in T98G and LN229 cell lines, respectively.) for 6 h. Cells were incubated for 72 h prior to imaging using a TCS SP8-Leica Microsystems confocal microscope (Leica, UK) with a 63x oil objective lens and subsequently analysed using LAS AF Lite Software (Leica, UK). Images are representative of three independent repeats. Fluorescence quantification of plasma membrane localised H-Ras in e LN229 and f T98G cells. The green fluorescence in three independent confocal images was quantified using ImageJ (National Institute of Health, USA). Results are mean ± SEM, n = 3