Tbc1d2

For mitochondria labeling, living cells were incubated with 5 µM MitoTracker red (Invitrogen) for 45 min at 37 °C. After this time, the cells were washed in PBS, fixed in 4% paraformaldehyde and counterstained with Hoechst 33258. For YAP, TBC1D2, LC3 and p62 staining, the cells were fixed with 4% paraformaldehyde, permeabilized by 0.5% (v/v) Triton X-100 and incubated for 1 h at 4 °C with specific primary antibodies: LC3 (Novus Biologicals; 1:100); p62 (Cell Signaling Technology; 1:100); YAP (Santa Cruz Biotechnology; 1:100); and TBC1D2 (Thermo Fisher Scientific; 1:100). AlexaFluor 488-conjugated anti-mouse IgG and AlexaFluor 594-conjugated anti-mouse IgG (both Invitrogen) were used as secondary antibodies. After washing, all samples were counterstained with Hoechst 33258 and then mounted in fluorescence mounting medium (Dako, Glostrup, Denmark). Images were acquired with intensified video microscopy (IVM) with an Olympus fluorescence microscope (Olympus Corporation, Milan, Italy) equipped with CoolLed pE-300-W (CoolLED Ltd., Andover, UK).