Ab267351

Hepatocytes were lysed with lysis buffer supplemented with phenyl methane sulfonyl fluoride (PMSF) as previously described
[17] (link). After centrifugation, protein concentrations in the extracts were analyzed using the BCA assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Proteins (30 μg from each extract) were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). Membranes were blocked with 5% defatted milk and then incubated with primary antibodies against DNMT1 (PA5-30581; Thermo Fisher Scientific), TFEB (ab267351; Abcam, Cambridge, USA), DNMT3a (ab2850; Abcam), DNMT3b (ab2851; Abcam), p62 (ab109012; Abcam), microtubule-associated protein 1 light chain 3 (LC3; ab192890; Abcam), and β-actin (ab8226; Abcam) respectively, followed by incubation with horseradish peroxidase (HRP)-labelled secondary antibody. Finally, enhanced chemiluminescence solution (Millipore) was used for detection of the indicated proteins, and immunoblot images were analysed with ChemiDoc (Bio-Rad, Hercules, USA).

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.

Hepatocytes were fixed with 4% paraformaldehyde for 20 min at room temperature (RT), permeabilized with 0.1% Triton on ice, and then blocked in PBS containing 5% bovine serum albumin (BSA) for 1 h, followed by incubation with a specific antibody against TFEB (ab267351; Abcam) overnight at 4°C. After that, Alexa Fluo-conjugated secondary antibody (Life Technologies, Waltham, USA) and DAPI were applied for 2 h at RT. Cells were then imaged with a BX51 laser confocal microscope (Olympus).