Glass bottomed culture dishes

For confocal laser scanning microscopy assays, DF1 cells grown in glass-bottomed culture dishes (NEST Biotechnology Co. LTD, Wuxi, China) were co-transfected with pCSGalNAcT2 and pGtVP2 as described above. Single transfections with 4 μg pCSGalNAcT2 or 4 μg pGtVP2 were performed as controls. At 36 h post-transfection, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 3% bovine serum albumin (BSA). Then, cells were incubated with the anti-FLAG monoclonal antibody and the anti-HA polyclonal antibody (Sigma) followed by addition of the corresponding FITC- or TRITC-conjugated secondary antibody for 1 h at room temperature. After washing three times with PBST containing 0.05% Tween-20, cells were stained with DAPI (Beyotime Institute of Biotechnology) for 10 min and analyzed using a Leica SP2 Confocal system (Leica Laser Technik, Heidelberg, Germany). Additionally, to determine whether the exogenous CSGalNAcT2 was expressed and located in the Golgi apparatus, we used confocal microscopy to evaluate the location of the exogenous CSGalNAcT2 and the endogenous GOLGA2 protein, a known marker for the Golgi apparatus, using GOLGA2 antibody (Sigma).

Transfected cells were plated in glass-bottomed culture dishes (NEST) for 24 h. They were fixed with 4% paraformaldehyde, and then washed with PBS. After that, cells were permeabilized with 0.1% Triton X-100 for 15 min, and then washed with PBS. After treating the cells with blocking buffer (Beyotime) for 30 min, they were incubated with anti-Flag, anti-Myc, E-cadherin and vimentin, at 4°C overnight. Then, fluorescence labelled secondary antibodies were applied, and following DAPI was counterstained for 1 h at room temperature. An anti-fluorescence quencher was added dropwise and fixed with a coverslip. Images were taken using a confocal microscope (LSM710, Zeiss).

NRVCs were seeded on glass-bottomed culture dishes (NEST Biotechnology, # 801002). For FAs visualization, BODIPY™ 558/568 C₁₂(Invitrogen, # D3835) was used following manufacturer’s instructions. Briefly, cells were incubated with complete medium containing 1 μM BODIPY™ 558/568 C₁₂ for 16 h. Before imaging, the medium was discarded and cells were wash with HBSS buffer for three time to remove the residual dye. For LD visualization, BODIPY™ 493/503 NHS Ester (Invitrogen, # D2191) was used following manufacturer’s instructions. Briefly, live cells or fixed cells were incubated with complete medium or PBS buffer containing 200 ng/ml BODIPY™ 493/503 NHS Ester for 1 min. Then the medium or PBS buffer was discarded and cells were wash with HBSS buffer for three time to remove the residual dye. All fluorescent images were obtained using a confocal laser-scanning microscope (Nikon A1 plus Confocal Microscope, Nikon, Japan). ImageJ colocalization finder was used to analyze the overlap coefficient between FFA and LD. ImageJ was used to calculate the area of LD.