Glass bottoms

HeLa cells were cultured in 24-well culture plates with glass bottoms (MatTek Corp., Ashland, MA, USA) and transfected with siRNA oligos targeting the indicated genes. Phase-contrast images were captured every 10 min for 24 h (12–36 h after transfection). Cells were imaged using an Axiovert 200M microscope equipped with EC Plan NEOFLUAR × 20 lens (Carl Zeiss). Images were acquired and processed using AxioVision 4.5 software (Carl Zeiss). The images were converted to JPEG format and exported to Adobe Photoshop.

MelJuSo cells were seeded on 35-mm-diameter imaging dishes with glass bottoms (MatTek) and transfected 24 h prior to the experiment. During imaging, the MelJuSo cells were kept at 37°C and 5% CO₂. To label acidic compartments, MelJuSo cells were incubated with LysoTracker^™ Red DND-99 for 30 min at 37°C and 5% CO₂ before being washed with 1x PBS and imaged, using a Zeiss LSM880 microscope equipped with a 63 × oil Plan Apo NA 1 objective. ACL cells were seeded on MatTek glass-bottom dishes coated with 20 μg/μL fibronectin (F2006-1MG Sigma) and transfected at least 4 days prior to the experiment. During imaging, the cells were kept at room temperature and 4.5% CO₂. To label acidic compartments, ACL cells were incubated with LysoTracker^™ Red DND-99 for 1 h at 15°C and 4.5% CO₂ and then imaged using a Zeiss LSM880 microscope equipped with a 63 × oil Plan Apo NA 1 objective. Mitochondrial staining was performed similarly, using MitoTracker Deep Red FM.