A0516

The distribution of β-catenin was measured as previously described [25 (link)]. After transfections, cells were fixated in 4% paraformaldehyde for 15 min and then processed with 0.1% Triton X-100 (ST795, Beyotime Institute of Biotechnology). After being sealed via goat serum, the slides were subjected to processing at 4 °C with anti-β-catenin (51067-2-AP; 1:200 dilution, Proteintech) overnight, and at 25 °C with secondary antibody (A0516; 1:200 dilution, Beyotime Institute of Biotechnology). Following nuclei-staining via DAPI, images were obtained using a microscope (Olumpus, Japan).

EdU staining was used to detect cell proliferation. Cells cultured on coverslips were fixed in paraformaldehyde (Beyotime, P0099) and permeabilized with 0.5% Triton X-100 (Beyotime, P0096). Subsequently, Edu staining kit (GeneCopoeia, A005) was carried out according to the manufacturer’s protocol. Cells were then visualized under a Leica microscope, as described above.
To analyze the subcellular location of TFEB and TFE3 transcription factors, the fixed and permeabilized cells were then incubated with monoclonal anti-TFEB or anti-TFE3 antibodies with or without anti-HPA antibody. The cells were then incubated with either anti-mouse antibody (Beyotime, A0428) or anti-rabbit antibody (Beyotime, A0468, or A0516) for 1 h. Subsequently, DNA staining (Beyotime, C1005) for 2 min and Leica confocal laser-scanning microscopy were performed as described above.
To assess autophagy, cells were transfected with a GFP-LC3 or GFP-RFP-LC3 plasmid, as mentioned above. After subsequent transfection with HPA wild-type or mutant plasmid, cells were subjected to confocal scanning microscopy.
To explore the effect on lysosome formation, Lyso-Tracker Red probe (Beyotime, C1046) staining was performed for 30 min at RT. Cells were then imaged under a Leica microscope, as described above.

Intestinal tissues were freshly isolated and fixed with 4% paraformaldehyde before paraffin embedding. After deparaffination and rehydration, the sections were stained with hematoxylin and eosin (H&E). For immunohistochemistry, tissues were treated with 0.01 M citrate buffer (pH 6.0) in a microwave and then blocked with blocking buffer (Beyotime, Shanghai, China) for 1 h. After incubation with primary antibodies at 4 °C overnight, sections were then immunostained using the SP method. For immunofluorescence staining, after incubation with the primary antibodies, the sections were incubated with Cy3-labeled Goat Anti-Rabbit IgG (1:1000, A0516, Beyotime, Shanghai, China) and counterstained with DAPI. The slides were cover slipped, and the stainings were observed by using a fluorescence microscope (Leica DM4/6B, Leica, Germany). The following primary antibodies were used: anti-VDR (1:200, 12550S, Cell Signaling Technology, Danvers, MA, USA), anti-Ki67 (1:200, ab16667, Abcam, Cambridge, MA, USA), anti-Sox9 (1:100, ab185230, Abcam, Cambridge, MA, USA), anti-Cleaved Caspase-3 (1:2000, 9664S, Cell Signaling Technology, Danvers, MA, USA) and anti-γH2AX (1:400, 9718T, Cell Signaling Technology, Danvers, MA, USA). At least 10 fields of view were captured, and 100 intact crypts were assessed per mouse. The crypt depth and immunopositive cells were quantified using Image J software.