UM-SCC-22A cells (50,000 per well) were plated on glass coverslips in a six-well plate for 18 hours. Cells were fixed and permeabilized using 4% paraformaldehyde (20 min) and 0.1% Triton X-100 in 1× PBS (phosphate-buffered saline) (pH 7.4) for 10 min. The cells were then blocked with 1% BSA (bovine serum albumin)/PBS (pH 7.4) for 1 hour. Cells were incubated for 18 hours at 4°C with antibodies specific for ceramide, LC3, Tom20, V5, GFP, FLAG, CerS1, GM130, or 11beta-HSD1 (1:50) in blocking solution, followed by Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibodies (1:1000) for 1 hour. Immunofluorescence was performed using a Leica TCS SP2 AOBS confocal microscope, an Olympus FV10i microscope with 543- and 488-nm channels for visualizing green and red fluorescence, or a Zeiss LSM 880 NLO Quasar confocal/multiphoton microscope with a Fast Airyscan super-resolution detector. Images were taken at 63× magnification. At least three random fields were selected for image quantification.

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