X090710 8

Cells were fixed in 4% paraformaldehyde at 4°C for 15 min. in the presence of a protein-blocking solution consisting of PBS supplemented with 5% normal goat serum (X090710-8, Agilent Technologies Inc., SantaClara, CA, USA). The cells were incubated overnight with anti-TFEB antibody (ab267351, Abcam plc.) in PBS at 4°C. The cells were washed extensively in PBS and incubated at room temperature for 30 min with a anti-rabbit IgG (H + L) antibody tagged with Alexa FluorTM 488 (Thermo Fisher Scientific, Inc.). The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; diluted 1:500, #5748, FUJIFILM Wako Pure Chemical) in PBS at room temperature for 30min. We obtained the fluorescence images using a Biorevo BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). The identification of TFEB migrated to nuclear was performed using ImageJ. First, the multicolor image was separated into TFEB- and DAPI-stained images. These images were converted to binarized images by thresholding, where a foreground pixel was assigned the maximum value of 255 and background pixels were assigned the minimum possible value of 80. The area where the TFEB and DAPI areas overlap is defined as the nuclear TFEB. The percentage of cells in the image with nuclear TFEB was calculated.

Formalin-fixed paraffin-embedded tissue sections (5 µm) were deparaffinized in xylene (3 times for 3 min each) and hydrated using 100% ethanol (twice for 5 min), 70% ethanol (5 min) and distilled water (twice for 3 min each). Antigen retrieval was done by heating the sections to 98 °C in citrate buffer pH 6.0 (Agilent-Dako, S236984) for 30 min. The slides were allowed to cool down for 30 min and were then rinsed in TBS twice for 5 min. Tissue sections were blocked with NGS (Agilent-Dako, X090710-8) for 30 min at room temperature followed by primary antibody incubation overnight at 4 °C. The sections were washed in TBS and incubated with peroxidase block (Agilent-Dako, K500711) for 30 min at room temperature. HRP-conjugated secondary antibody was then applied for 30 min at room temperature, followed by TBS washes and DAB incubation for 5–10 min. The sections were counterstained with haematoxylin according to a standard procedure. A list of the antibodies used is provided in Supplementary Table 1.

Cells were fixed with 4% PFA at 4 °C for 5 min and permeabilized with 0.1% Triton X-100 at room temperature for 20 min in the presence of a protein-blocking solution consisting of PBS supplemented with 5% normal goat serum (#X090710-8, Agilent Technologies Inc., Santa Clara, CA, USA). The cells were incubated overnight with primary antibodies in PBS at 4 °C. The cells were washed extensively in PBS and incubated at room temperature for 30 min with a secondary antibody. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; diluted 1:500, #5748, FUJIFILM Wako Pure Chemical) in PBS at room temperature for 30 min. To prevent fading during microscopy, the cells were mounted in DakoCytomation fluorescent mounting medium (#S302380-2, Agilent Technologies Inc.). Immunofluorescence images were visualized and recorded using a Biorevo BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). Quantifications of PBs carried out in triplicated experiments by blinded independent researchers, who chose five to seven fields in each session.