Lsm710 system

Cells were seeded into 8-well μ-Slides (Ibidi) at a density of 80,000 cells/well. After overnight incubation, cells were washed with pre-warmed PBS, fixed with 4% formaldehyde solution for 15 min at room temperature, washed, and then blocked with complete blocking solution (0.5% BSA, 0.1% TritonX-100, 5% goat serum in sterile PBS) for one hour at room temperature. Next, samples were incubated overnight at 4 °C with the relevant primary antibodies (anti-E-Cadherin antibody (ab11512-Abcam), anti-Vimentin Antibody (V9) (sc-6260-SantaCruz), anti-gamma H2A.X (phospho S139) antibody (ab11174-Abcam) anti-cytokeratin 8 (ab53280-Abcam) and anti-cytokeratin 14 (ab7800-Abcam). After incubation, the cells were washed with PBS, and the secondary antibodies (Alexa Flour 488, Alexa Fluor 546, Alexa Fluor 555) were added in complete blocking solution, followed by incubation for two hours at room temperature. Nuclei were labeled with DAPI. Imaging was carried out using a ZEISS LSM-710 system (Carl Zeiss microscopy Gmbh, Jena, Germany) with a 40×/1.4 Plan-Apochromat oil immersion objective. Images were processed with ZEN (Carl Zeiss microscopy Gmbh, Jena, Germany). γ-H2AX foci were counted with FindFoci, an automated ImageJ plugin [96 (link)].

Cellular internalization and localization of antibodies in cultured cells were detected by confocal microscopy22 (link)23 (link). Briefly, cells (5 × 10⁴) that were grown on 12-mm diameter coverslips in 24-well culture plates were treated with indicated antibodies, as specified in the figure legends. Internalized antibodies were detected with Alexa488-conjugated goat anti-human IgG antibody (Invitrogen, A11013, dilution 1:500) for 1 h at 25 °C. Ras proteins were detected with rabbit anti-Ras antibody (Abcam, ab108602, dilution 1:100) and subsequently with TRITC-conjugated anti-rabbit antibody (Sigma-Aldrich, T6778, dilution 1:250) for 1 h at 25 °C. The nucleus was stained with Hoechst 33342 in PBS for 5 min at 25 °C. After mounting the coverslips onto glass slides with Perma Fluor aqueous mounting medium (Thermo Scientific, TA-030-FM), center-focused single z-section images were obtained on a Zeiss LSM710 system with ZEN software (Carl Zeiss).

MCF-7 cells were seeded in a complete cell medium to 24-well plates, which contained cover glasses (thickness 1, Assistant, Karl Hecht GmbH & Co KG, Sondheim/Rhön, Germany). After one day, the treatment was performed in a serum-free medium for different time points (5, 15, 30 and 60 s as well as 5, 10, 30 and 60 min). Afterwards, cells were washed twice with phosphate buffered saline (PBS) and fixed by 4% paraformaldehyde for 20 min at 37 °C. To stain the nuclei, the samples were washed three times with PBS and incubated for 15 min with 4′,6-diamidine-2-phenylindole dihydrochloride (DAPI, 0.2 µg/mL, dissolved in PBS, Sigma-Aldrich Kft). After washing, cover glasses were mounted to microscopy slides (VWR International, Debrecen, Hungary) by Mowiol^® 4–88 mounting medium (Sigma-Aldrich Kft). Confocal microscopy studies were performed on a Zeiss LSM 710 system (Carl Zeiss Microscopy GmbH, Jena, Germany) with a 40X oil objective and ZEN Lite (Carl Zeiss Microscopy GmbH) software was used for image processing.

Cytospins were prepared with freshly dissociated cells. After spinning, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 1% Triton X-100 in PBS for 10 min. Cells were blocked with 10% of normal goat serum for 30 min at 37°C, and then incubated with a primary antibody against CD44 (1:100; Santa Cruz Biotechnology), CD47 (1:100, Abcam) and KRT14 (1:100, Santa Cruz Biotechnology) for 1 h in humid atmosphere. After rising, cells were incubated with secondary goat anti-mouse IgG-Alexa Fluor 594-conjugated (1:100, Invitrogen), and the nuclei were counterstained with Hoechst (5 μg/mL). Negative controls were prepared by omitting staining with the primary antibody. Images were acquired in a confocal microscopy Zeiss LSM710 system (Carl Zeiss AG) using a × 63 1.4 NA oil immersion lens. The quantification of fluorescence intensities was performed using the NIH ImageJ 1.47v analysis software. Regions were drawn around each fluorescent cells and in a region without fluorescent objects for background subtraction. The corrected total cell fluorescence (CTCF) was determined using the following formula: CTCF = integrated intensity - (area for the selected cell × mean background). Data is represented as the mean of fluorescence intensity (MFI).

SH-SY5Y cells were seeded into coverslips (thickness 1, Assistent®, Karl Hecht GmbH & Co KG, Sondheim vor der Rhön, Germany) containing 24-well plates (Sarstedt) for microscopy studies at a density of 7.5 x 10⁴ cells/well, in 1 mL complete DMEM medium. The following day, cells were treated with Cf-HSV peptides at a concentration of 25 µM (diluted in serum free DMEM medium) for 3 h. Lysosomes were stained with Lysotracker Deep Red (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Nuclei were visualised with Hoechst 33342 (Thermo Fisher Scientific, 0.2 µg/mL). After each step, cells were washed three times with serum-free medium. Following the staining and washing steps, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, Budapest, Hungary), the solution was prepared in-house, washed with PBS (pH = 7.4). The coverslips were mounted with Mowiol 4–88 (Sigma-Aldrich, Budapest, Hungary) to microscopy slides. Imaging was performed by a Zeiss LSM-710 system (Carl Zeiss microscopy GmbH, Oberkochen, Germany) with a 40×/1.4 Plan-Apochromat oil immersion objective using lasers with excitation maxima 405, 488 and 633 nm for detecting Hoechst, Cf-conjugated peptides and LysoTracker, respectively. Images were processed with ZEN (Carl Zeiss microscopy GmbH).

For immunofluorescence staining, sections were blocked with PBS 2% goat serum 0.3% Triton X-100 for one hour at RT, and then incubated with rabbit monoclonal anti-Ki-67 (Abcam, 1:200), rabbit monoclonal anti-cleaved caspase 3 (Cell Signaling Technology, 1:200), or mouse monoclonal anti-EBP50 (BD Biosciences, 1:50) for one hour at 37°C. After washing in PBS, slides were incubated for one hour at RT with goat anti-mouse or anti-rabbit Alexa Fluor 488- or 546-conjugated antibodies (Invitrogen, 1:800). During the last ten minutes of incubation 4', 6-diamidino-2-phenylindole (DAPI, Invitrogen, 1:100) was added. Sections were analyzed by confocal laser-scanning microscope (Zeiss LSM-710 system, Carl Zeiss Microimaging GmbH) or by fluorescence microscope (Olympus BX61, Olympus Inc.).