Figure 3. LDLR contributes to cellular entry of TcdA^1–1832.

a. The absence of LDLR expression in three LDLR^−/− HeLa cell lines was confirmed by immunoblot analysis. Actin served as a loading control. The experiments were repeated three times independently with similar results.

b. The sensitivities of three LDLR^−/− HeLa cell lines to TcdA^1–1832 and TcdB^1–1830 were quantified using the cytopathic cell-rounding assay and normalized to the levels of WT HeLa cells (n=6). Each data point was also shown as triangle in the bar graph.

c. The sensitivities of HeLa WT and LDLR^−/− cells to full-length TcdA were evaluated using the cytopathic cell-rounding assays (n=6). The percentage of rounded cells were quantified, plotted, and fitted.

d. Ectopic expression of a mouse Ldlr in LDLR^−/− (#4) cells restored the sensitivity of these cells to TcdA^1–1832 and resulted in cell rounding under our assay conditions (2 nM, 4 h). Green fluorescent protein (GFP) was co-transfected to mark transfected cells. Representative fluorescence images of transfected cells were shown on the left side, while the percentage of rounded cells were quantified and shown on the right side (n=6). Each data point was also shown as triangle in the bar graph.

e. Pre-incubation of the ecto-domain of LDLR (residues 22–788, 400 nM) with TcdA^1–1832 (2 nM, 4 h) protected HeLa cells from the toxin and prevented cell-rounding. The experiments were repeated three times independently with similar results.

f. Pre-incubation of RAP (4 μM) in culture medium further protected LDLR^−/− (#4) cells from TcdA^1–1832 (10 nM) as measured by the cell rounding assay over time (n=6).

For d and e, scale bar represents 50 μm.

Error bar represents mean ± s.d.. Experiments were repeated two times independently with similar results.