Fig. 7: Cellular uptake and intoxication by dual-modified C3 proteins.

a Fluorescence confocal microscopy showing uptake of C3 protein conjugates, C3-Cy3-Cy5 and C3-RGD-Cy5 into A549 cells treated with 300 nM of the respective C3 variants for 24 h and co-stained with NucBlue. Each experiment was repeated independently two times with similar results. DAPI channel at 405 nm, Cy5 channel at 649 nm and Cy3 channel at 554 nm. Scale bar: 10 μm. b–d A549 cells were treated with 100 nM or 300 nM of C3, C3-Cy3-Cy5 or C3-RGD-Cy5 at 37 °C for 5 h or left untreated (Ctrl). b Phase contrast microscopic images of intoxicated A549 cells. Scale bar: 100 μm. c Quantification of cells showing C3-morphology as percentage of total cell number. Values are given as mean of duplicates within one experiment. d Intracellular Rho ADP-ribosylation status of cells after treatment for 8 h. Non-modified Rho in each cell lysate was biotin-labelled in the sequential ADP-ribosylation reaction by C3. Biotin-ADP-ribosylated Rho was detected in a Western blot by streptavidin-peroxidase conjugate, where a weak signal correlates with a high ADP-ribosyltransferase activity of C3 conjugates in living cells. Hsp90 served as loading control (n = 4). Detailed experimental information is provided in SI. c, d Source data are provided as a Source Data file.