Panel A shows fluorescence microscopy images and an intensity profile demonstrating EPHA2 and ASO colocalization. Panel B shows density plots comparing nuclear-proximal and -distal ASO-positive vesicle fluorescence intensities. Panel C shows microscopy images and bar graphs quantifying ASO internalization in EPHA2-deficient cells. Panel D shows immunoblot analysis measuring EPHA2 phosphorylation following cET-ASO treatment over time. Panel E shows dose-response curves comparing Kras messenger RNA reduction between EPHA2 variants. Panel F shows fluorescence microscopy and bar graphs quantifying RAB17 colocalization with ASO vesicles. Panel G shows dose-response curves comparing Kras messenger RNA reduction after Rab17 knockout. Panel H shows immunoblot analysis of MEK and ERK phosphorylation after Rab17 knockout. Panel I shows dose-response curves comparing Kras messenger RNA reduction between EPHA2 variants. Panel J shows immunoblot analysis comparing MEK and ERK phosphorylation in EPHA2 variants. Panel K shows a bar graph comparing relative cell proliferation following cET-ASO treatment. Panel L shows density plots comparing ASO vesicle distributions after BafA1 treatment durations.
EPHA2-mediated trafficking and nuclear capture dictates cET-ASO Kras efficacy. (A) Internalization for 16 h of cET-ASOKras (red) in KPC cells showing overlap of cET-ASOKras and EPHA2 (green) in internalized vesicles (white arrows). (B) Quantification and binning of cET-ASOKras-positive vesicle-nucleus distance into proximal (0–1 µm) or distal (>1 µm) categories. Density plots show ASO fluorescence intensities per ASO-positive vesicle expressed as log-transformed pseudo-counts. n = 3 independent experiments. Paired t test. (C) cET-ASOKras-positive intracellular vesicle area per cell and relative intensity per vesicle in either KPC Epha2+/+ (blue bars) or Epha2−/− (magenta bars) cells after ASO treatment, n = 16 cells. (D) EPHA2 phospho-Ser897 in KPC cells with and without treatment with 1 μM cET-ASOKras. (E)Kras mRNA expression after 72 h of cET-ASOKras treatment in EPHA2 WT rescue (Epha2−/−+ EPHA2WT, gray) or EPHA2S897A mutant rescue (Epha2−/−+ EPHA2S897, salmon) KPC cells; data are mean ± SEM, n = 4 independent experiments. (F) Overlap between cET-ASOKras (green) and RAB17-positive vesicles (red) in KPC cells; data are mean ± SEM, n = 4 independent experiments. (G)Kras mRNA expression in Epha2+/+ nontargeting-CRISPR (gray line) and Rab17-CRISPRKO KPC cells (blue line) after 72 h of cET-ASOKras treatment; data are mean ± SEM, n = 6 independent experiments. (H) MEK1/2 and ERK1/2 induction in Epha2+/+ nontargeting or Rab17-CRISPRKO KPC cells after 72 h of cET-ASOKras treatment. Vinculin is a loading control. (I)Kras mRNA expression after 72 h of treatment with cET-ASOKras in EPHA2 WT rescue (Epha2−/−+ EPHA2WT, gray) or EPHA2 NLS mutant rescue (Epha2−/−+ EPHA2NLS, magenta) KPC cells; data are mean ± SEM, n = 7 independent experiments. (J) MEK1/2 and ERK1/2 activation in Epha2−/−+ EPHA2WT and Epha2−/−+ EPHA2NLS KPC cells after 72 h of cET-ASOKras. Vinculin is a loading control. (K) Cell proliferation after cET-ASOKras (96 h) treatment in either Epha2−/−+ EPHA2WT (blue bar) or Epha2−/−+ EPHA2NLS (magenta bar) KPC cells; data are mean ± SEM, n = 4 independent experiments. TWA, Sidak for all panels. (L) Density plots of ASO-positive vesicles (vesicle number per area of cytoplasm) in cells treated either with vehicle or the indicated concentrations of bafilomycinA1 in combination with either vehicle or ASO (5 μM) for either 4 or 16 h. Data are expressed as log10-transformed, n = 3 independent experiments. All conditions are compared with vehicle-ASO–treated cells, ANOVA, and Tukey post hoc test. Source data are available for this figure: SourceData F2. TWA, two-way ANOVA.