Primary cultures of Human Fibroblasts (HFs) were grown on coverslips and processed as previously reported [14 (link)]. Cells were then incubated with the primary mouse monoclonal anti-LC3 (1:100 in PBS, 5F10 Nanotools, Teningen, Germany) for 1 h at 25 °C and then with a goat anti-mouse IgG-Alexa Fluor 488 (1:200 in PBS, Life Technologies, Carlsbad, CA, USA) for 30 min at 25 °C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (1:1000 in PBS; Sigma-Aldrich, St Louis, MO, USA). Coverslips were finally mounted with Mowiol solution (Sigma-Aldrich, St Louis, MO, USA). Fluorescence signals were analyzed by scanning cells in a series of sequential sections with an ApoTome System (Zeiss), image analysis was performed by the Axiovision software (Zeiss), and images were obtained by 3D reconstruction of all the serial optical sections. Quantitative analysis of LC3-positive dots per cell was performed using ImageJ software [21 (link)], analyzing 100 cells for each sample in 5 different microscopy fields from 3 different experiments (n = 3). Results are shown as means ± standard deviation (SD). Student’s t-test was performed, and significance levels have been defined as p < 0.05.
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