FIGURE 2.

Effect of sdAb-Fcs on DiI-LDL uptake by HepG2 cells and their LDLR cell surface levels. HepG2 cells were incubated for 5 h in the absence or presence of 0.08 μm (5 μg/ml) PCSK9 mixed with increasing concentrations of sdAb-Fcs. DiI-LDL (5 μg/ml final) was added during the last 2 h of incubation. The % DiI-LDL uptake shown here is normalized to the number of cells per well and calibrated to the % uptake in control cells (in absence of PCSK9) (A). A similar experiment was performed with evolocumab (B). In both A and B, the data represent averaged values ± S.E. for at least three independent experiments, with each comprising three independent samples per condition. HepG2 cells were incubated for 4 h without (no PCSK9) or with 0.05 μm (3 μg/ml) PCSK9 (+PCSK9) in the absence or presence of each sdAb-Fc (1.2 μm). LDLR immunocytochemistry with Alexa 555-labeled secondary antibodies was performed, and nuclei were stained with DAPI. Scale bar, 20 μm (C). In two separate experiments, LDLR labeling was quantified based on the average pixel values of 10 fields calibrated to the number of nuclei per field. The numbers (bottom left corner) are expressed as % of the value obtained in the absence of PCSK9 (no PCSK9) and represent averaged pixel values of the two independent experiments. *, p < 0.05; **, p < 0.005; ***, p < 0.0005. p values were obtained from Student's t tests, except for some conditions in A, for which a two-way ANOVA test was more appropriate.