Journal: PLoS ONE

Article Title: Coordinate Autophagy and mTOR Pathway Inhibition Enhances Cell Death in Melanoma

doi: 10.1371/journal.pone.0055096

Figure Lengend Snippet: (A) Melanoma cell lines were transiently transfected with an EGFP-LC3 expressing plasmid and grown in normal medium. 24 hours after transfection, the cells were fixed and the autophagosomes in the cells were visualized by the presence of LC3 puncta under fluorescence microscopy, with the percentages of cells showing LC3 puncta (mean ± SD) indicated. All tested cell lines showed positive punctation indicating high basal autophagy. (B) Western blot shows that melanoma cell lines have high basal LC3-II and p62 protein levels. Upon HBSS induced starvation, increased cleavage of LC3-I to LC3-II indicative of autophagy induction and decreased or unchanged p62 levels were seen. Extracts from autophagy competent and autophagy deficient iBMK cells served as positive and negative controls. (C) Immunofluorescence staining for endogenous LC3 on a human melanoma tissue microarray. Punctate LC3 localization for autophagosomes is indicated by red arrows (top panel). The percentages of specimens with punctate LC3 staining in malignant, metastatic and benign nevus groups are indicated in the table (bottom panel). (D) Western blot showed decreased expression levels of Atg7 (top panel) and impaired clonogenic survival (bottom panel) in response to lentiviral shRNA knockdown of the essential autophagy regulator Atg7.

Article Snippet: Human Malignant Melanoma Tissue Microarray (US Biomax, Inc) slides, or xenograft tumor paraffin slides were deparaffinized, hydrated and incubated with primary antibody against LC3 (Nanotools, 1∶100 dilution) for 15 min at room temperature followed by FITC tagged anti-mouse (Jackson Immuno Research) antibody, then mounted and visualized by epifluorescence microscopy.

Techniques: Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Immunofluorescence, Staining, Microarray, shRNA