Plan apochromat 63

Octpep-1 tagged with fluorescein at the N-terminus was purchased from GenScript (Leiden, The Netherlands). MM96L cells were grown in coverslips and treated with 200 µg/mL Octpep-1 tagged with fluorescein at 37 °C for 48 h. Additionally, mitochondrial co-staining was performed by incubation with 100 nM MitoTracker Red (excitation, 581 nm; emission, 644 nm) for 30 min at 37 °C followed by washes with PBS and 30 min fixation with 4% PFA at room temperature. Fluoroshield Mounting Medium with DAPI (4′,6-diamidino-2-phenylindole) was used to stain the nucleus and preserve fluorescence in the fixed samples. Images of the emitted fluorescence were acquired at 300, 50, and 100 ms of exposure for green, red, and blue fluorescence, respectively, using a Leica DMIL microscope with a Plan- Apochromat 63 × 1.4 NA oil immersion objective. Analysis was completed using ImageJ-Fiji software (National Institutes of Health, Tennessee, USA). Data are expressed as corrected total cell fluorescence using the following equation [(Cell Area × Mean Cellular Fluorescence) − (Cell Area × Mean Background fluorescence)].

Transfected cells were processed for immunofluorescence as described^{50 (link)}. Briefly, cells were fixed for 10 min with 3% paraformaldehyde at room temperature, permeabilized with 0.1% Triton-X100 in PBS, incubated with primary antibodies, washed, incubated with secondary antibodies, and mounted with ProLong Gold antifade mounting solution (Thermo Fisher Scientific). Cells were observed with epifluorescence microscopes: Zeiss AxioImager M2m equipped with AxioCam color CCD camera, with Plan-Neofluar 40× lens (NA 0.75) and Plan-Apochromat 63× lens (NA 1.4). Confocal images were acquired at a Perkin Elmer UltraVIEW spinning disk confocal microscope with EM-CCD camera and Plan-Apochromat 63× lens (NA 1.4); or at a Leica TCS SP5 or TCS SP8 SMD FLIM laser scanning confocal microscope equipped with HC PLAPO CS2 63x lens (NA 1.4). For quantitative analysis of the projected cell area, transfected cells were randomly imaged at a wide field microscope (Zeiss Axio Observer.Z1 equipped with Hamamatsu 9100 - 02 EM CCD Camera). For evaluation of the subcellular localization of transfected and of endogenous proteins, confocal images were visually analyzed. For quantification, 2–4 independent experiments per condition were analyzed using Fiji^{51 (link)}.