Control and WIPI1 shRNA (pGIPZ)-expressing human melanocytes were fixed in 2% paraformaldehyde for 1 h. Coverslips were rinsed with PBS, and cells were permeabilized with 0.4% saponin (primary melanocytes) and blocked in 1% BSA with 0.1% Tween 20 for 1 h. Cells were incubated with anti-LC3B antibody (Invitrogen) and then labeled with secondary antibodies conjugated with Alexa Fluor 594 (Invitrogen). For each clone, the cytosolic fluorescence intensity per unit area in 10 cells was quantified using ImageJ, and the data were then normalized to control shRNA-expressing melanocytes. Statistical significance was determined using a Student's t test. MNT-1 cells expressing control and WIPI1 shRNAs (pLKO) were immunostained by anti-β-Catenin (Cell Signaling Technology, Inc.), EEA1 (Cell Signaling Technology, Inc.), LAMP2 (Abcam), and Alexa Fluor 488 (Invitrogen) using the same method described above except that 0.2% Triton X-100 was used for permeabilization. For β-Catenin, the nuclear fluorescence intensities of 100 cells per unit area in each treatment was quantified using ImageJ, and the data were then normalized to vehicle-treated control shRNA-expressing MNT-1 cells. Statistical significance was determined using a Student's t test. For EEA1 and LAMP2, total fluorescence intensities per unit area in 10 cells was quantified using ImageJ, and the data were then normalized to the value of control shRNA-expressing MNT-1 cells, and statistical significance was determined using a Student's t test. Colocalization studies were performed in MNT-1 parent cells using rabbit LC3B antibody (Invitrogen) with mouse PMEL17 or TYRP1 (Santa Cruz Biotechnology Inc.) or using mouse p62-SQSTM1 antibody (BD Biosciences) and rabbit PMEL17 (Sigma) or TYRP1 (Abcam) followed by secondary antibodies conjugated to Alexa Flour 594 or 488 (Invitrogen). Coverslips were mounted in a solution containing DAPI. Confocal images were acquired using a LSM 510 META confocal multiphoton microscope or a Nikon Ti fluorescence microscope as indicated. The three-dimensional projected images were processed as 12-bit images by LSM Image Browser (v4.0, Zeiss). Full paper
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