Labtec chambers

The pore-forming activity of St I (20 nM) was measured by adding free Alexa 488 or Alexa 555 or fluorescent labeled Dextrans (100 nM) in phosphate-buffered saline (PBS) to the vesicles and gently mixing. The experiments were performed in LabTec chambers (Nalge Nunc International, Rochester, NY, USA), previously blocked with bovine serum albumin (10 mg/mL), on a LSM710 microscope with a C-Apochromat 40× water immersion objective using as excitation light Ar-ion (488 nm) and HeNe lasers (543 nm and 633 nm). Images were processed automatically using a software implemented in MATLAB (The MathWorks, Inc., Natick, MA, USA) [41 (link)]. The percentage of GUVs filling at 30 min was calculated as follows: $$\mathrm{Permeabilized} \mathrm{GUVs} (\%)=\frac{{\mathrm{F}}_{30 min}^{in}-{\mathrm{F}}_0}{{\mathrm{F}}_{30 min}^{out}-{\mathrm{F}}_0}\ast 100$$ where ${\mathrm{F}}_{30 min}^{in}$ and ${\mathrm{F}}_{30 min}^{out}$ are the average fluorescence intensities inside and outside GUVs at 30 min, respectively, and ${\mathrm{F}}_0$ is the background fluorescence at 30 min. Below the threshold of 20% of filling the GUVs were considered non-permeabilized, and above 80% filling GUVs were considered totally permeabilized as previously suggested [39 (link)].

24 h after transfection of hTERT-GFP constructs, HeLa cells grown on LabTec-chambers (Nunc) were transferred to CO₂-independent medium (Gibco). FLIP-experiments were carried out at 37°C in a temperature controlled chamber attached to a confocal microscope (510-Meta, Carl Zeiss, Jena, Germany) equipped with an argon laser and a 63x Plan-Neofluar 1.3NA water-corrected objective. For analysis of nuclear import during 146 seconds, a nuclear region was subjected to 180 bleach intervals with the argon laser set to 100%. After each interval, the fluorescence emission was collected during a period of 154 ms at 2% laser intensity with the pinhole set to 2.0 airy units. Signal intensities were analyzed from cytoplasmic regions of a bleached (F_bc) and a neighboring unbleached cell as a reference (F_rc), and a background region (F_bg). Cytoplasmic fluorescence intensities for each time point were expressed as F(t) = (F_bc–F_bg)/(F_rc–F_bg), normalizing the pre-bleach fluorescence to 1. 10–20 cells were analyzed per condition.