Dapi fluoromount g solution

For cell staining, A375 cells expressing shTIM (shMock, sh1, sh2) and shTIPIN (shMock, sh1, sh2) were incubated with the primary antibody. One day later, Alexa Fluor 488-labeled or Alexa Fluor 568-labeled secondary antibodies (Thermo Scientific) were added. The DAPI-Fluoromount-G^® solution (Electron Microscopy Sciences, Hatfield, PA, USA) was added for nuclei staining of the cells. Fluorescence-labeled proteins were analyzed using the Zeiss fluorescent microscope with the apotome attachment and Zeiss software and Axiocam cameras (Carl Zeiss Microscopy GmbH, Deutschland).

For mitochondrial staining, HCT-116^p53+/+ and HCT-116^p53−/− live cells were first incubated with 0.3 μM MitoTracker Red CMXRos (Invitrogen, Eugene, Oregon, USA) for 30 min at 37 °C in 5% CO₂. For co-localization and for mitochondrial staining, colon cancer cell lines were fixed with 3.7% formaldehyde, and then permeabilized in 300 μl of 0.5% Triton X-100. In general for immunostaining, after blocking the cover slips in 10% BSA for 1 h, the primary antibody was added overnight. The day after, Alexa Fluor 488-labeled or Alexa Fluor 568-labeled secondary antibodies (Thermo Scientific) were added (see Table S2). The DAPI-Fluoromount-G® solution (Electron Microscopy Sciences, Hatfield, PA, USA) was added for nuclei staining of the cells. Fluorescence-labeled proteins were analyzed using the Zeiss Fluorescent microscopy with apotome attachment and the Zeiss software and Axiocam cameras (Carl Zeiss Microscopy GmbH, Deutschland).