Inhibition of clathrin-mediated endocytosis significantly blocks uptake of siRNA lipoplexes but not functional siRNA delivery. BSC cells were treated with chlorpromazine (Chlorp, 10 μg/mL) or cytochalasin D (CytoD, 100 μM) for 1 h followed by siRNA lipoplex transfection for 4 h in the presence of the inhibitors. After washing, siRNA lipoplex uptake was measured by flow cytometry (see for details). Transfected cells were cultured for another 20 h before CyB transcript level was measured by real-time PCR. (A) Uptake of Cy5-siRNA lipoplexes in the absence or presence of inhibitors. (B) Relative levels of CyB transcript in the presence or absence of inhibitors as normalized to the level of untreated cells. (C) Relative levels of CyB transcript in BSC cells that expressed the wild-type (WT) or K44A dynamin. BSC cells that stably express either the WT or K44A dynamin were transfected with siRNA lipoplexes for 4 h, washed, cultured for 20 h, and then assayed for CyB transcript level. Data shown are mean ± SD (n = 4). Representative data from one of three to five experiments are shown.

Functional delivery of siRNA lipoplexes is not dependent on caveolin- or lipid-raft-mediated endocytosis or macropinocytosis. (A) Relative levels of CyB transcript in BSC cells that express the wild-type (WT) or the mutant caveolins. BSC cells that stably expressed either the wild-type or one of the mutant caveolins were transfected with siRNA lipoplexes for 4 h, washed and cultured for 20 h. The level of CyB transcript was quantified. (B, C) Effect of PDMP (B) or amiloride (C) on functional siRNA delivery. BSC cells were treated with PDMP (10 μM) or amiloride (3 mM) for 1 h, followed by siRNA lipoplex transfection for 4 h, washed, and cultured for 20 h. The level of CyB transcript was quantified. (D) Uptake of Cy5 labeled siRNA lipoplexes in the absence or presence of inhibitors as quantified by flow cytometry. Representative data are shown as mean ± SD (n = 4). P values between selected samples are shown. Representative data from one of three experiments are shown.

Uptake of the bulk of siRNA lipoplexes and functional siRNA delivery are not correlated. (A) Imaging analysis of siRNA lipoplexes inside the cells. Cy5-siRNA lipoplexes were incubated with BSC cells for the indicated length of time, washed, and cultured for the indicated length of time before fixation and imaging analysis. Cy5-siRNA lipoplexes were red and nuclei were stained blue. Representative images at the indicated times are shown. Scale bar, 10 μm. (B) siRNA lipoplexes colocalize with endolysosomes. BSC cells were transfected with Cy5-siRNA lipoplexes for the indicated length of time in the presence of LysoSensor. Live cell imaging was carried out at different time points after transfection. The number of intracellular siRNA complexes and the number of siRNA complexes that overlapped with LysoSensor staining were enumerated. Percentages of intracellular siRNA complexes that colocalize with LysoSensor are shown at the indicated time points. (C) Kinetics of target mRNA knockdown. BSC cells were transfected with CyB-specific siRNA lipoplexes for 2 or 4 h, washed, were used for RNA isolation immediately. Some 4 h transfected cells were cultured for another 2 or 20 h before RNA isolation. The levels of CyB transcript were quantified by real-time PCR and normalized to that in nontransfected cells. Percentages of target mRNA level are shown as mean ± SD. P values between selected samples are indicated. Representative data from one of three to five experiments are shown.

Cholesterol depletion inhibits functional siRNA delivery. (A) Uptake of siRNA lipoplexes in the absence or presence of either nystatin (100 μg/mL) or filipin (10 μg/mL) as measured by flow cytometry. (B) Relative CyB transcript level in the presence or absence of the inhibitors. Representative data are shown as mean ± SD (n = 4). P values between selected samples are shown. Representative data from one of three experiments are shown.

Effect of lowing temperature and inhibiting ATP biosynthesis on functional delivery of siRNA lipoplexes. (A) Uptake of siRNA lipoplexes in the absence or presence of deoxy-glucose (40 mM) and sodium azide (40 mM). (B) Relative level of CyB transcript in the presence or absence of ATP biosynthesis inhibitors. (C) Effect of temperature on knockdown of target transcript. Representative data are shown as mean ± SD (n = 3 or 4). R² value of exponential fit is shown. Representative data from one of three experiments are shown.