Gated stimulated emission depletion microscopy gsted

One hundred thousand MCF7 cells were seeded onto coverslips in 12-well plates. When cells were attached, mDIVI1 and vehicle treatments were performed in triplicate. After 2 days and 5 days, mitochondria were labeled by incubating cells for 15 min at 37° C with 25 nM of MitoTracker DeepRed (M22426, Invitrogen) diluted in PBS (D8662, Sigma). Then, cells were washed with PBS, fixed in 2% paraformaldehyde (28908, Thermo Scientific) in PBS for 30 minutes at room temperature and mounted with ProLong^® Gold Antifade Mountant reagent with DAPI (P36935, Invitrogen, Inc.). Immunofluorescence pictures were taken in a Leica gated Stimulated Emission Depletion Microscopy (gSTED) with additional confocal and multi-photon illumination (room rg106).

1.5 × 10⁵ hTERT-BJ1 fibroblasts per well were seeded in coverslips in 12 well plates. When cells were attached, drug treatments were added for 72 h in triplicate. Cells were fixed with 2% paraformaldehyde (28908, Thermo Scientific) in PBS for 20 minutes at RT, permeabilised in −20°C-cold methanol for 5 minutes at 4°C, quenched with 50 mM NH4Cl in PBS for 10 minutes, rinsed and blocked with IF buffer consisting of 1% BSA (A3608, Sigma) plus 0.1% Tween 20 (P9416, Sigma) in PBS for 1h. Cells were then incubated for 1h with the MCT4 primary antibody (sc-50329, Santa Cruz). The fluorescent secondary antibody was added for 30 minutes. Cells were counterstained with DAPI and samples were mounted using Prolong Gold anti-fade reagent (P36934, Invitrogen). Immunofluorescence pictures were taken in a Leica gated Stimulated Emission Depletion Microscopy (gSTED) with additional confocal and multi-photon illumination (room rg106).