Lsm710 confocal laser scanning microscopes

Rapamycin and BafA1 were obtained from Santa Cruz Biotechnology and Focus Biomolecules, respectively. MitoTracker Green FM was purchased from Thermo Fisher Scientific. Etoposide was purchased from Sigma-Aldrich. Other chemicals, including CCCP, were purchased from Nacalai Tesque. Fluorescence images were obtained using LSM710 confocal laser-scanning microscopes (Zeiss). For CLEM observation, a JEM-1400Plus transmission electron microscope (JEOL) was used with a CCD camera (EM-14830RUBY2; JEOL).

Cells cultured on coverslips were fixed with 4% PFA, followed by permeabilization with 50 µg/mL digitonin (for spreading assays and staining of FAs) or with MeOH at −20 °C (for staining of flotillins). Thereafter, the cells were incubated with the primary antibody in 1% BSA or Alexa Fluor 488-conjugated phalloidin (Molecular Probes, Eugene, OR, USA), washed, incubated with the secondary antibody and mounted in GelMount (Biomeda, Foster City, CA, USA). The samples were analyzed with a Zeiss LSM 510 or LSM 710 Confocal Laser Scanning Microscopes (Carl Zeiss, Oberkochen, Germany).
To visualize FAs, the coverslips were coated with either collagen or fibronectin (both 10 µg/mL). The number of FAs was determined in HeLa cells seeded on collagen. For counting FAs, the size of the cells was determined by measuring their length and width. Focal adhesions were counted only in cells exhibiting a certain size range (mean of each experiment ±25%). The experiment was repeated five times, and 50 cells were analyzed in total for each condition.