Plan apochromat 63 1.4 na m27 oil immersion objective

The immunostained cells were examined on an LSM 880 + Airyscan system (Carl Zeiss) with a Plan-Apochromat 63×/1.4 NA M27 oil immersion objective using immersion oil (#518F, Carl Zeiss) at room temperature. The microscope was operated on the ZEN 2012 software platform (Carl Zeiss). Detailed sample preparation and observation condition were described in Supplemental Information. After calculation of processing for the super-resolution, the images were processed in the ZEN 2012 software and ImageJ 1.47v. Three-dimensional reconstruction of GFP-NLS-TDP25 was performed in the Imaris x64 7.4.2 software (Bitplane, Zurich, Switzerland).

Cells were grown on eight-well Lab-Tek II Chamber Slides, treated as indicated and fixed with 4% paraformaldehyde for 10 min at room temperature. Blocking was performed with 3% bovine serum albumin in PBS + 0.02% saponin for 1 h at room temperature. Immunostainings were performed on dilution of primary antibodies in blocking solution and overnight incubation at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at room temperature. After three more washes, coverslips were finally mounted in VECTASHIELD mounting medium with 4,6-diamidino-2-phenylindole and analysed using LSM 800 or LSM 880+ Airyscan systems (Carl Zeiss), with a Plan-Apochromat ×63/1.4 NA M27 oil immersion objective using immersion oil (catalogue no. 518F, Carl Zeiss) at room temperature. The microscopes were operated on the ZEN 2013 software platform (Carl Zeiss). After calculation of processing for the airyscan, images were processed in the ImageJ v.1.47. Mander’s colocalization coefficient was calculated using JACoP ImageJ Plugin^{61 (link)}.

Immunofluorescence experiments were performed as previously described^{39 (link)}. Briefly, cells were grown on 8-well Lab-Tek II-Chamber Slides, treated as indicated, and fixed with 4% parafolmaldehyde (PFA) for 10 min at RT. For endogenous TFEB staining, cells were permeabilized with 0.1% Triton X-100 for 5 min, followed by blocking with 3% bovine serum albumin in PBS + 0.02% saponin for 1 h at RT. Immunostainings were performed upon dilution of primary antibodies in blocking solution and overnight incubation at 4 °C, followed by three washes and secondary antibody incubation in blocking solution for 1 h at RT. After additional three washes, coverslips were finally mounted in VECTASHIELD® mounting medium with DAPI and analyzed using LSM 700 or LSM 800 with a Plan-Apochromat ×63/1.4 NA M27 oil immersion objective using immersion oil (#518F, Carl Zeiss) at room temperature. The microscopes were operated on the ZEN 2013 software platform (Carl Zeiss).