Figure 4: Macropinocytosis-mediated cellular uptake of CaP-rHDL in a Ras activation-dependent manner.

(a) Quantitative analysis of the cellular uptake of DiI-CaP-rHDL in astrocytes, C6, U87, U251, MIA PaCa-2, BxPC-3, SW-480 and Caco-2 cells after incubation at 37 °C for 3 h at the DMPC concentration of 5 μg ml⁻¹. (b) The level of Ras-GTP in astrocytes, C6, U87, U251, MIA PaCa-2, BxPC-3, SW-480 and Caco-2 cells evaluated via a Ras activation assay kit. (c) A linear regression was established to model the relationship between the cellular uptake of CaP-rHDL and the intracellular Ras-GTP level in the different cell types (R²=0.8687). (d) Knockdown of general all Ras led to a reduction in the cellular uptake of DiI-CaP-rHDL. Ras knockdown was achieved following the application of a shRNA expression lentivirus system. (e) Knockdown of each Ras-subtype led to a reduction in the cellular uptake of both the marker of macropinocytosis TMR-dextran and DiI-CaP-rHDL. Ras knockdown was achieved following the application of shRNA expression lentivirus systems. A comparable level of transfection efficiency between the various shRNAs was indicated by monitoring the intensity of green fluorescent protein (GFP), which is concomitantly expressed with each shRNA. Compared with that in those cells transfected with a negative control shRNA (NC-shRNA), the cellular uptake of TMR-dextran (red) and DiI-CaP-rHDL (red) were reduced in cells transfected with KRas-specific shRNA, NRas-specific shRNA and HRas-specific shRNA. Scale bar, 10 μm. For a,b and d, data represent mean±s.d. (n=3). The significance of the differences between two groups (*P<0.05, **P<0.01, ***P<0.001) was evaluated by two-tailed Student’s t-test.