Bodipy fl c5 ceramide d 3521

Cells were cultured on glass coverslips, were treated as indicated, and were fixed with 4% paraformaldehyde for 30 min. After the coverslips were blocked with 10% normal goat serum for 1 h at room temperature, the cells were incubated with an anti-LC3 antibody or an anti-LAMP2 antibody (1:100, Santa Cruz Biotechnology) overnight at 4 °C. After an incubation with the corresponding secondary antibodies for 1 h at 37 °C, the cells were stained with BODIPY (FL C5-ceramide D-3521, Life Technologies, Invitrogen, Grand Island, NY, USA; stock solution: 1 mg/ml in ethanol, working solution: 10 μg/ml diluted in PBS) in the dark at room temperature for 20 min. A subsequent staining was performed with 1.0 ml of 0.5 μg/ml Hoechst 33258 for 5 min at 37 °C in the dark and the cells were examined with laser scanning confocal microscopy.

Human osteosarcoma cell lines 143B, HOS-MNNG, and MG63 were purchased from the American Type Culture Collection (ATCC) and utilized according to the manufacturer’s instructions. ZOSM was obtained from the First Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China). Mycoplasma contamination was excluded via PCR, and cell line identification was confirmed using short tandem repeat (STR) profiles. Osteosarcoma cells were cultured in DMEM (Corning, USA) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Myriocin and MG132 were purchased from MedChemExpress. BODIPY™ FL C₅-Ceramide (D3521), BODIPY™ FL C5-Sphingomyelin (D3522), and Alexa Fluor 647-conjugated CTB (V34404) were purchased from Invitrogen (USA). The following antibodies were used: anti-β-actin (A5316) from Sigma-Aldrich and anti-CD151 (96262) and anti-c-myc (18583) from Cell Signaling Technology (USA). Anti-SPTLC1 (ab23287) antibody was purchased from Abcam (Cambridge, UK).