*5.6. Coupling of Maleimide-Labeled Cargo to Cys\_C3botE174Q*

The modular Cys\_C3botE174Q system was loaded with maleimide-labeled cargo by incubation at 4 ◦C for 2 h. For mDL488 (Thermo Scientific, Waltham, MA, USA), Cys\_C3botE174Q and cargo were mixed at a 1:1 molar ratio. Due to the steric hindrance of PEG for mPEG\_FITC (Nanocs, Boston, MA, USA), a 1:50 molar ratio (Cys\_C3botE174Q to cargo) was needed. mC2I was prepared by labelling C2I at 4 ◦C for 2 h with a 10-fold molar excess of MBS (Thermo Scientific, Waltham, MA, USA) followed by removal of unbound MBS with Zeba Spin Desalting Columns (7 kDa molecular weight cutoff, Thermo Scientific, Waltham, MA, USA). For generation of mC2I-Cys\_C3botE174Q, Cys\_C3botE174Q and mC2I were mixed at a 1:1 molar ratio. Subsequent to coupling of the cargo, free maleimidelabeled small-molecule cargo (mDL488 and mPEG\_FITC) was removed by two rounds of buffer exchange via the Zeba Spin Desalting Columns with 7 kDa molecular weight cutoff protocol. Finally, the concentration of loaded transporters was determined via SDS-PAGE by comparison to a BSA standard.

#### *5.7. Flow Cytometry*

Human-monocyte-derived macrophages were detached with 1 mM EDTA/PBS for 20 min at 37 ◦C, while lymphocytes were harvested by centrifugation (1300 rpm for 10 min). Per sample, either 2 × <sup>10</sup><sup>5</sup> macrophages or lymphocytes were incubated with His\_eGFP or His\_eGFP\_C3botE174Q, or left untreated for 20 min at 37 ◦C. Afterwards, cells were washed with FACS buffer (1% FCS (Biochrom, Berlin, Germany) and 0.1% sodium azide (VWR, Radnor, PA, USA) in PBS (Gibco-Life Technologies, Carlsbad, CA, USA) and centrifuged for 10 min at 1300 rpm. The supernatant was discarded, and cells were incubated directly before measurement for 1 min with 50 μg/mL trypan blue to quench extracellular eGFP

signals as described in [40]. Intracellular fluorescence was detected using a FACSCalibur™ flow cytometer (BD Biosciences, Heidelberg, Germany)**.** Data analysis was performed using Flowing Software 2.5.1 (Turku Bioscience, Turku, Finland).

#### *5.8. Phase Contrast Microscopy*

Cells were seeded in 96-well microtiter (Corning Incorporated, Corning, NY, USA) plates and incubated with the indicated test substance at 37 ◦C and 5% CO2 on the next day. One phase contrast image was taken per well (three wells for each treatment) with a LEICA DMi1 microscope connected to a MC 170 HD camera (both Leica Microsystems, Wetzlar, Germany). Representative images are depicted with scale bars.

#### *5.9. Cell Viability and Proliferation Assay*

Cells were seeded in a 96-well microtiter plate and treated as indicated. After the indicated incubation time, 10 μL CellTiter 96 AQueous One solution was added, containing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium (MTS). Cells were incubated further for 1–2 h, and the absorbance at 492 nm was measured in a plate reader.

#### *5.10. STED Super-Resolution Microscopy*

STED microscopy was performed as described in [23,40]. A total of 10<sup>5</sup> monocytederived macrophages were seeded per well in an 8-well μ-slide with a glass bottom (ibidi GmbH, Gräfelfing, Germany). The cells were incubated with 250 nM of the indicated protein ( His\_eGFP, His\_eGFP\_C3bot, or His\_eGFP\_C3botE174Q) for 30 min at 37 ◦C. Afterwards, the cells were washed twice with cold PBS and fixated with 3.2% paraformaldehyde in PBS (32% PFA aqueous solution, Electron Microscopy Sciences, Hatfield, PA, USA) for 20 min at RT. After washing the cells three times with PBS, the cells were permeabilized and blocked in 3% BSA and 0.3% TritonX-100 in PBS for 2 h. The samples were incubated overnight with 1 μg/mL of primary rabbit anti-EEA1 antibody (Thermo Scientific, Waltham, MA, USA) and 0.5 μg/mL Atto594-conjugated GFP-booster nanobody (Chromotek, Planegg-Martinsried, Germany) in 1:10 diluted blocking solution at 4 ◦C. The cells were washed three times and incubated with 1 μg/mL of the secondary Atto647N-conjugated goat anti-rabbit antibody (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1:10 diluted blocking solution. Unbound antibodies were removed by washing the samples three times with PBS. Before imaging, PBS was exchanged with 2,2 -thiodiethanol (97% solution in PBS, pH 7.5). A self-build dual-color 3D STED microscope [53] was used for image recording with an average power of 0.8 μW for each excitation beam and 1.3 mW for each depletion beam. The pixel size was 12.5 nm, and images were captured with 300 μs dwell time and approximately 150 counts as a typical peak photon number. The recorded pictures were analyzed with ImageJ (v1.52n, National Institute of Health, Bethesda, MD, USA). A Gaussian blur σ = 1 pixel and >20 count intensity threshold was applied for better visualization.

The eGFP signals and their co-localization with EEA1 signals were automatically quantified by a self-written search algorithm in Python 3.7. The algorithm loaded respective raw image file pairs, i.e., one image file containing Atto594-conjugated GFP-booster nanobody signal intensities and the belonging second image file representing signal intensities for EEA1. On both image files, a Gaussian blur σ = 1 pixel was applied to reduce background noise and to smoothen signals for later automated search. Next, a threshold of >35 counts was set for eGFP signal intensities and >50 counts for EEA1 signal intensities to further eliminate unwanted background. After thresholding, the algorithm horizontally searched for eGFP signals. Whenever a signal was found horizontally, the respective vertical coordinate was searched for. Each coordinate pair was saved for later use and counted. Thereby, for each donor, the mean signal number per image was calculated and averaged for five individual blood donors (n = 5). After every pixel was analyzed with regard to eGFP signals, the saved eGFP coordinate pairs were loaded to be compared with the image file containing EEA1 signal intensities. In more detail, within a radius of 250 nm (based on an

estimated endosome diameter of 500 nm) from the previously found eGFP coordinate pair, the algorithm searched for EEA1 signals, indicating an EEA1-associated eGFP signal. Such co-localizing signals were successively counted. EEA1 signals above a radius of 250 nm were cut off as non-co-localizing eGFP signals. For each donor, the mean percentage of co-localizing eGFP signals was calculated (co-localizing eGFP signals divided by the total number of eGFP signals) and averaged for five individual blood donors (n = 5).

#### *5.11. Immunofluorescence Staining for Confocal or Epifluorescence Microscopy*

Cells were incubated as indicated at 37 ◦C in 5% CO2. Afterwards, cells were washed twice and fixed (4% PFA, Sigma-Aldrich, St. Louis, MO, USA). For MHC class II staining, cells were incubated with 2% BSA in PBS and labeled with anti-HLA-DR antibody (1:200, L243, Leinco, St. Louis, MO, USA) for 30 min at RT. After three washing steps with PBS, MHC class II was detected by Cy5-conjugated goat anti-mouse antibody (1:250, Dianova, Hamburg, Germany). If indicated, cell nuclei were stained either with DAPI (1:200, Sigma-Aldrich, St. Louis, MO, USA) diluted in 1% BSA and 0.1% Triton X-100 in PBS, or with 5 μg/mL Hoechst33342 in PBS for 10 min at RT. Confocal images were acquired by using the inverted laser scanning confocal microscope LSM 710 (Zeiss, Oberkochen, Germany). Epifluorescence microscopic images were recorded using the iMIC digital microscope (FEI, Munich, Germany). Images were processed using ImageJ software (v1.51n, National Institute of Health, Bethesda, MD, USA).

#### *5.12. SDS-PAGE and Western Blotting*

For protein separation depending on molecular weight, SDS-PAGE was used with 12.5% acrylamide gels. Subsequently to electrophoresis, the proteins were transferred onto a nitrocellulose membrane by using semi-dry blotting, which was controlled by Ponceau S (AppliChem GmbH, Darmstadt, Germany) staining. Unspecific binding to the membrane was blocked by incubation in 5% skim milk powder diluted in PBS-T (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.8 mM KH2PO4, 0.1% Tween20; pH 7.4) for 1 h at RT. After washing with PBS-T, the membrane was incubated with the indicated antibody or streptavidin– peroxidase conjugate (1:5000; Sigma-Aldrich, St. Louis, MO, USA) diluted in PBS-T for 1 h at RT. For eGFP detection, 1:10,000 diluted anti-GFP antibody (ab290, Abcam, Cambridge, GBR) was used. HSP90 was detected with 1:500 diluted HSP90 α/β antibody (F-8, Santa Cruz Biotechnology, Dallas, TX, USA), while for EEA1-detection, 1:1000 diluted rabbit EEA1 polyclonal antibody (PA1-063A, Thermo Fisher Scientific, Waltham, MA, USA) was used. Unbound antibodies/proteins were removed with three washing steps with PBS-T for 5 min at RT on an orbital shaker. For the detection of eGFP and EEA1, 1:2500 diluted mouse anti-rabbit IgG-HRP (sc-2357, Santa Cruz Biotechnology, Dallas, TX, USA) was used. For detection of HSP90, 1:2500 diluted m-IgGκ BP-HRP (sc-516102, Santa Cruz Biotechnology, Dallas, TX, USA) was used. The peroxidase-labeled antibodies/proteins were detected with Pierce ECL Western blotting substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA Health Care, Mortsel, BEL).

#### *5.13. Sequential ADP-Ribosylation Assay*

Cells were seeded in 24-well microtiter plates and treated as indicated at 37 ◦C and 5% CO2. Extracellular toxins were removed by washing the cells two times with PBS. The medium was removed, and the samples were frozen at −20 ◦C and thawed in ADPribosylation buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, 5 mM MgCl2, cOmplete (1:50, freshly added); pH 7.5) to lyse the cells. The samples were collected, and 5 pmol fresh C3bot and 6-biotin-17-NAD<sup>+</sup> (10 μM) were added in excess. The sequential ADP-ribosylation reactions at 37 ◦C were started and stopped at the same time (after 30 min). Laemmli buffer (0.3 M Tris-HCl, 10% SDS, 37.5% glycerol, 0.4 mM bromophenol blue) was added, and the samples were heat denatured. Notably, in this sequential ADP-ribosylation reaction, only the non-ADP-ribosylated Rho can be biotin-labeled from 6-biotin-17-NAD+. Hence, in SDS-PAGE and Western blotting (see Section 5.12), the non-ADP-ribosylated Rho in intact

cells is detected with streptavidin–peroxidase conjugate. For densitometric analysis of the detected protein bands, ImageJ software (v1.51n, National Institute of Health, Bethesda, MD, USA) was used. Importantly, weak signals detected in this assay indicate strong toxin activity in intact cells.

#### *5.14. Digitonin-Based Cell Fractionation Assay*

A total of 10<sup>6</sup> U-DCS cells were seeded on a 24-well microtiter plate. After two days, the cells were incubated with the respective proteins (His\_eGFP\_C3bot, His\_eGFP\_C3botE174Q, and His\_eGFP) with indicated concentrations and time points at 37 ◦C. Subsequently, the cells were carefully washed twice with PBS and then incubated with digitonin (20 μg/mL in PBS) for 5 min at RT. Thereby, the cell membrane was permeabilized, and the cytosol left the cells through the previously formed pores during incubation for 25 min at 4 ◦C on ice. In this process, the cells were divided into two fractions: the supernatant (cytosol-only fraction) and the solid cellular portion (membrane fraction) containing cellular organelles, vesicles, cell membranes, and the remaining cytosolic proteins that did not flow out through the pores. The fluorescence of eGFP was analyzed in both fractions using a microplate reader at 488 nm excitation and 510 nm emission. SDS-PAGE and Western blot analysis (see Section 5.12) were performed to ensure clean separation of both fractions. The early endosomal marker EEA1 was only detectable in the membrane fraction. Cytosolic HSP90 can be detected in both fractions since it is not possible to force complete outflow of all cytosolic proteins. The presence of His\_eGFP and His\_eGFP-labeled proteins was analyzed in the respective cell fractions with the GFP antibody.

#### *5.15. Data Analysis and Visualization*

The depicted data points are provided as mean ± standard deviation (± SD) with corresponding sample size (n). A two-tailed unpaired Student's *t*-test was used to compare the two treatment groups. The following significance levels were defined: not significant (ns) *p* > 0.05, \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001. Diagrams were generated by using GraphPad Prism (version 9.1.2 GraphPad Software, San Diego, CA, USA). Figures were assembled in Inkscape (version 0.92, Free Software Foundation, Boston, MA, USA).

**Supplementary Materials:** The following supporting information can be downloaded at https://www. mdpi.com/article/10.3390/toxins14100711/s1, Table S1. Quantification of the mean eGFP signals per image given for the individual human blood donors. Table S2. Co-localization analysis of eGFP and EEA1. Table S3. Quantification of the cytosol fraction from the digitonin-based cell-fractionation assay. Table S4. Quantification of the membrane fraction from the digitonin-based cell-fractionation assay. Figure S1. Cell-type selective uptake of His\_eGFP\_C3botE174Q into primary human macrophages compared to lymphocytes ex vivo. Figure S2. Quantification of eGFP-positive macrophages from Figure 2b. Figure S3. Detection of the eGFP-labeled C3bot variants in SDS-PAGE and Western blotting. Figure S4. The modular thiol–maleimide system enabled attachment of cargo molecules to Cys\_C3botE174Q. Figure S5. Internalization of mPEG\_FITC into macrophages and DCs was strongly enhanced by coupling to Cys\_C3botE174Q. Figure S6. The cell viability/proliferation of U-DCS cells was not or only minimally effected by the transported mC2I.

**Author Contributions:** Conceptualization, M.F., S.F. and H.B.; Funding acquisition, S.F., J.M., S.S. and H.B.; Investigation, M.F., M.S., R.N. and F.W.; Project administration, H.B.; Supervision, S.F., J.M., S.S. and H.B.; Visualization, M.F.; Writing—original draft, M.F., M.S. and R.N.; Writing—review & editing, M.F., M.S., R.N., F.W., S.F., J.M., S.S. and H.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the German Research Foundation (DFG) as part of the CRC 1279 (A01 and C02)—Project number 316249678—SFB 1279 and part of the CRC 1149 (A05)—Project number 251293561—SFB 1149 together with a CRC 1149 start-up grant (Stephan Fischer). Maximilian Fellermann, Mia Stemmer, and Reiner Noschka are members of the International Graduate School in Molecular Medicine Ulm (IGradU) and grateful thank the IGradU for its support.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study is available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the study design; in collection, analyses, or interpretation of data; in writing of the manuscript, or in the decision to publish the results.

#### **References**

