Material & methods

6.2.5.1 Antibodies and reagents

Rabbit monoclonal anti-β-catenin (ab32572) was obtained from Abcam (Cambridge, UK), the mouse monoclonal anti-cortactin clone 4F11 from Millipore (Billerica, MA, USA).

Alexa Fluor-labelled secondary goat anti-rabbit or anti-mouse IgG antibodies were obtained from Molecular Probes (Eugene, OR, USA). Alexa Fluor-labelled phalloidin was from Invitrogen (Merelbeke, Belgium). The synthetic peptide GGGY-propargylglycine (GGGY-PAG) was chemically synthesized by Peptide Specialty Laboratories GmbH (Heidelberg, Germany). pEVOL-pAzF was a gift from Prof. Dr. Peter Schultz (Scripps Research Institute, Cincinnati, OH, USA) (Addgene plasmid #31186;

http://n2t.net/addgene:31186; RRID:Addgene_31186)²²⁵. Sortase A pentamutant in pET29 was a gift from Prof. Dr. David Liu (Harvard University, Cambridge, MA, USA) (Addgene plasmid #75144; http://n2t.net/addgene:75144; RRID:Addgene_75144)⁴⁵². 6.2.5.2 cDNA cloning

Nanobodies were cloned into a pMECS or pHEN6 vector, as described before^{47, 215}. The nanobodies were subcloned by means of a Cold Fusion Cloning Kit (System Biosciences, Mountain View, CA, USA). To add the sortag to a nanobody, the first subcloning was performed into pMECS vector by using the following primers: forward 5’-GATGTGCAGCT GCAGGAGTCTGGAGGAGG-3’ and reverse 5’-CTAGTGCGGCCGCGCCACCGGTTTCCGGAAGC GCGCTGCCTGAGGAGACGGTGACCTGGGT-3’. For amber stop codon introduction, cloning of the nanobodies in the pHEN6 vector was done using the following primers: forward pHEN6 5’-CCAGGTGCAGCTGCAGGAGTCTGGGGGAGGATT-3’ and reverse pHEN6 5’-GTCAC CGTCTCCTCAGGAGGAAGCGGTGGCTAGACCGGTCATCATCACCATC-3’.

6.2.5.3 Recombinant nanobody and sortase A pentamutant production

The production and purification of the nanobodies, was performed as described previously⁴⁷. E. coli WK6 cells were transformed with nanobodies in pMECS/pHEN6 vector. They were grown at 37 °C in TB with 100 μg/ml ampicillin (Duchefa, Haarlem, the Netherlands) and if the pEVOL-pAzF was co-transformed, with 34 μg/ml chloramphenicol (Duchefa) and 1 mM pAzF. After induction with 0.2% (w/v) arabinose (Sigma-Aldrich, St.

Louis, MO, USA) and 1 mM IPTG (Biosynth, Staad, Switzerland), incubation was performed at 28 °C overnight. French press was used to obtain total cell lysis followed by short sonication. Next, an IMAC purification was performed as described before⁴⁷. Briefly, the recombinant His6-tagged nanobodies were purified from E. coli WK6 cells by

binding to Co²⁺-chelating resins (Takara Bio Inc., France) and were recovered from the beads with standard elution buffer (50 mM NaH2PO4, 500 mM NaCl, 250 mM imidazol, pH 8.0), the potassium phosphate elution buffer (100 mM potassium phosphate, 100 mM KCl, 250 mM imidazole, pH 7.4) or the HEPES elution buffer (50 mM HEPES (Duchefa), 50 mM NaCl (Duchefa), 250 mM imidazole, pH 8.0).

Sortase A pentamutant in pET29 was transformed in E. Coli BL21 cells and grown at 37 °C in LB with kanamycin. After induction with IPTG, they were incubated at 20 °C overnight.

French press was used to obtain total lysis followed by short sonication. Next an IMAC purification was performed as mentioned above.

6.2.5.4 Sortase A reaction

The nanobodies were dialyzed into a sortase compatible buffer (50 mM Tris-HCl (Duchefa Biochemie, Haarlem, The Netherlands), 150 mM NaCl, pH 8.0). Subsequently, the sortase reaction was performed at 30 °C for 90 min. The reaction mixture (total volume 100 µl) consists of 50 µM peptide, 4 µM sortase A pentamutant, 20 µM nanobody in sortase reaction buffer (50 mM Tris-HCl, 150 mM NaCl and 10 mM CaCl2 (Sigma-Aldrich, St. Louis, MO, USA), pH 8.0). Thereafter, immobilized metal affinity chromatography (IMAC) purification was used to remove His6-tagged proteins still present in the mixture.

6.2.5.5 CuAAC after sortase: test experiment for optimisation – benefit of HEPES

After sortase reaction, the nanobodies were used directly or were diafiltered through an Amicon Ultra-0.5 Centrifugal Filter Unit (MWCO 3kDa) (Sigma-Aldrich) according to the manufacturer’s instructions, into a buffer with lower Cl^- ions concentration (10 mM HEPES-NaOH, 50 mM NaCl, pH 8). Subsequently, Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC) was performed. Two tubes were prepared of which the first tube contained CuSO4 (20 mM stock solution) (Merck, Overijse, Belgium), tris(3-hydroxypropyltriazolylmethyl)amine (THTPA) (50 mM stock solution) (Sigma-Aldrich) and sodium ascorbate (100 mM stock solution) (Sigma-Aldrich) and the second contained HEPES (100 mM stock solution), an alkyne-bearing nanobody and an azide-Alexa Fluor AF488 (5 mM stock solution) (Jena Bioscience, Jena, Germany). Bringing the content of both tubes together, the final concentrations (Buffer 1 at Table 2) were 0.1 mM CuSO4, 0.5 mM THTPA, 5 mM sodium ascorbate, 10 mM HEPES-NaOH, 3 µM nanobody and 6 µM azide-AF488. This was incubated overnight at 33 °C and analysed on sodium dodecyl

sulphate – polyacrylamide gel electrophoresis (SDS-PAGE) and with a FastGene FAS-digi Geldoc device (NIPPON genetics, Dueren, Germany).

6.2.5.6 CuAAC after sortase: test experiment for optimisation – benefit of THPTA or sodium ascorbate

After sortase reaction, the nanobodies were diafiltered through an Amicon Ultra-0.5 Centrifugal Filter Unit (MWCO 3kDa) (Sigma-Aldrich) according to the manufacturer’s instructions into a low Cl^- ions solution (10 mM HEPES-NaOH, 50 mM NaCl, pH 8).

Subsequently, CuAAC was performed. Two tubes were prepared of which the first tube contained CuSO4 (20 mM stock solution) (Merck), THTPA (50 mM stock solution) (Sigma-Aldrich) and sodium ascorbate (100 mM stock solution) (Sigma-(Sigma-Aldrich) and the second contained HEPES (100 mM stock solution), an alkyne-bearing nanobody and an azide-AF488 (5 mM stock solution) (Jena Bioscience). Bringing the content of both tubes together started the reaction. The end concentration in this mixture was determined by the conditions in the experiment. For the following compounds, the final concentrations remained constant at 0.1 mM CuSO4, 50 mM HEPES-NaOH, 5 µM nanobody and 10 µM azide-AF488. THTPA and sodium ascorbate were different in the different conditions as can be seen in Table 2. The mixtures were incubated overnight at 33 °C and analysed by SDS-PAGE and with a FastGene FAS-digi Geldoc device (NIPPON genetics).

6.2.5.7 CuAAC after pAzF incorporation: test experiment for optimisation – time point determination

After the pAzF incorporation (see supplementary material), nanobodies were dialyzed against a HEPES buffer (50 mM HEPES-NaOH, 50 mM NaCl, pH 7.4) to make them compatible with the CuAAC reaction. Two tubes were prepared (one with CuSO4 (20 mM stock solution), THTPA (50 mM stock solution) and sodium ascorbate (100 mM stock solution), the other with HEPES (100 mM stock solution), pAzF containing nanobody and alkyne-AF488 (5 mM stock solution) (Jena Bioscience)) and mixed gently to obtain final concentrations of 0.1 mM CuSO4, 1 mM THTPA, 7.5 mM sodium ascorbate, 50 mM HEPES-NaOH, 30 µM nanobody and 60 µM alkyne-AF488. At each time point (0, 5, 15, 20, 30, 45, 60 and 75 min) during the CuAAC reaction, a 3 µl sample out of the total reaction mixture volume of 400 µl was removed and analysed by SDS-PAGE and with a FastGene FAS-digi Geldoc device (NIPPON genetics).

6.2.5.8 CuAAC to obtain fluorescently labelled nanobodies - final protocol After the pAzF incorporation, nanobodies were dialyzed against HEPES buffer (50 mM HEPES-NaOH, 50 mM NaCl, pH 7.4) to make them compatible with the CuAAC reaction.

Before the start of CuAAC, two tubes were prepared. The first tube contains CuSO4 (20 mM stock solution) (Merck), THTPA (50 mM stock solution) (Sigma-Aldrich) and sodium ascorbate (100 mM stock solution) (Sigma-Aldrich), while the second tube contained HEPES (100 mM stock solution), a pAzF containing nanobody and an alkyne-AF488 (5 mM stock solution) (Jena Bioscience). After mixing both tubes, the final concentrations in this mixture are 0.1 mM CuSO4, 1 mM THTPA, 7.5 mM sodium ascorbate, 50 mM HEPES-NaOH, 30 µM nanobody and 60 µM alkyne-AF488. This was incubated for 60 min at 33

°C. Thereafter, an EDTA solution (20 mM HEPES-NaOH, 500 µM EDTA (Sigma-Aldrich), pH 7.4) was used to complex the Cu²⁺ ions. Their removal from the mixture was achieved by diafiltration using an Amicon Ultra-0.5 Centrifugal Filter Unit (MWCO 3kDa) (Sigma-Aldrich). At least 3 times the reaction volume of the EDTA solution was used during the diafiltration. This was followed by a PD Spintrap™ G-25 (Sigma-Aldrich) used according to the manufacturer’s instructions. The product was analysed by SDS-PAGE and a FastGene FAS-digi Geldoc device(NIPPON genetics), and protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA).

6.2.5.9 Immunocytochemistry and microscopy

The immunocytochemistry was performed as described before³⁸⁹. Cells were seeded on a 100 µg/ml gelatine (Sigma-Aldrich) or 50 µg/ml rat tail type I collagen (BD Biosciences, Franklin Lakes, NJ, USA) coated coverslip. In case of a 4% paraformaldehyde (PFA) fixation, cells were fixed using 4% PFA, permeabilized using 0.2% TritonX-100 (Sigma-Aldrich) for 5 minutes and neutralized and blocked in 20 mM glycine (Sigma-(Sigma-Aldrich) for 20 minutes. In case of a methanol fixation, cells were fixed and permeabilised with 100%

ice-cold methanol for 5 minutes and blocked in 20 mM glycine (Sigma-Aldrich) for 60 minutes. After the blocking steps, an incubation with primary antibodies or labelled nanobody (1 h at 37 °C) was performed and if necessary followed by Alexa Fluor-conjugated secondary antibodies (30 min at room temperature). DAPI (0.4 µg/ml; Sigma) and Alexa Fluor labelled phalloidin (Invitrogen) were used to stain nuclei and actin filaments, respectively. Cells were mounted using VectaShield (Vector Laboratories, Burlingame, CA, USA). For imaging, an Olympus IX81 FluoView 1000 confocal laser scanning microscope (UPlanSApo x60 1.35-NA UplanSApo objective; Olympus, Tokyo, Japan) with FluoView FV1000 software (Olympus) was used at room temperature.

6.2.5.10 Photoporation

The photoporation was performed as described in more detail elsewhere⁶⁷. Briefly, a µ-slide (µ-Slide Angiogenesis, ibidi, Beloeil, Belgium) was seeded with 5000 HeLa cells per well one day in advance. Before laser treatment, cells were incubated with graphene quantum dots-PEG suspended in cell culture medium for 30 min. The fluorescently labelled nanobody was dispersed in Dulbecco's phosphate-buffered saline (DPBS) at a concentration of approximately 40 μg/mL. 10 μL nanobody solution was added into each well before the photoporation laser treatment. The sample was scanned through the photoporation laser beam (20 Hz pulse frequency) using an electronic microscope stage (HLD117, Prior Scientific, USA). The scanning speed was 2.1 mm/s, and the distance between subsequent lines was 0.1 mm to ensure that each cell received a single laser pulse. The total scanning time per well was 2 min. The fluorophore solution was then removed and the cells were gently washed with DPBS and supplemented with fresh cell culture medium. Samples were analysed using a spinning disk confocal microscope (Nikon eclipse Ti-e inverted microscope, Nikon, Japan) equipped with an MLC 400 B laser box (Agilent technologies, Santa Clara, CA, USA), a Yokogawa CSU-22 Spinning Disk scanner (Andor Technology, Belfast, UK) and an iXon ultra EMCCD camera (Andor). A 60×/1.4 oil immersion objective lens (CFI Plan Apo VC 60 × oil, Nikon, Japan) was used for imaging.

6.2.5.11 Cell culture

HNSCC61 and HeLa cells were maintained at 37 °C in a humidified 5% CO2 incubator. All were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum, 10 µg/mL streptomycin and 10 IU/mL penicillin. In case of HNSCC61 cells, an extra 0.4 µg/mL hydrocortisone (Sigma-Aldrich) was added and 20% fetal bovine serum was used.

6.2.5.12 Generation of β-catenin nanobodies

A cDNA fragment covering the C-terminal half of human catenin (amino acids 398-end) was cloned into the pTYB12 prokaryotic expression vector and expression/purification was done as described for other constructs described above. A llama was immunized with this protein fragment by a subcutaneous injection on days 0, 14, 28 and 35.

Anticoagulated blood was collected on day 39 for lymphocyte preparation. By extracting total RNA from peripheral blood lymphocytes, a nanobody library was constructed and screened for the presence of antigen-specific nanobodies by phage panning. The

obtained nanobodies were subcloned into the phagemid vector pMECS. They are currently licensed to Gulliver Biomed (www.gulliverbiomed.com).