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

Journal: Nature Genetics

Article Title: Loss of NECTIN1 triggers melanoma dissemination upon local IGF1 depletion

doi: 10.1038/s41588-022-01191-z

Figure Lengend Snippet: ( a ) Dot plot representing NECTIN1 mRNA expression and linear copy number in the TCGA cohort of human cutaneous melanomas (363 samples). Spearman correlation: r = 0.38, p = 3.8E-14. Four outliers with high NECTIN1 expression were removed from the graph but retained in the analysis. ( b ) Box-and-whisker plot representing NECTIN1 mRNA expression depending on NECTIN1 copy-number status (363 samples). Min-max, 5 and 95 percentiles, median. Brown-Forsythe ANOVA. Unpaired two-tailed Welch’s t-test: deep deletion vs diploid: p = 0.0013, shallow deletion vs diploid: p = 0.0006. Four outliers with high NECTIN1 expression were removed from the graph but retained in the analysis. ( c ) Representative histology and scoring system used for the evaluation of NECTIN1 expression by immunohistochemistry, related to Fig. . 0 = Negative (<1% tumor cells immunoreactive); 1= weak and incomplete staining in < 10% of cells, 2 = weak/moderate heterogeneous staining in > 10% of cells, or 3 = strong and complete homogenous staining in > 10% of cells. Scale bar: 50 μm. Representative of 3 independent tissue-microarrays. ( d ) Distribution of NECTIN1 staining level by immunohistochemistry in 253 tissue sections of human primary melanomas or metastases. Correlation was measured by Chi-square test. ( e ) Dot plot representing NECTIN1 and CDH1 mRNA expression in the TCGA cohort of human cutaneous melanomas (363 samples). Spearman correlation: r = −0.08, p = 0.107. Four outliers with high NECTIN1 expression were removed from the graph but retained in the analysis. ( f ) Immunofluorescence analysis of E-cadherin (green) and NECTIN1 (red) on tissue sections of 4 different human melanomas exhibiting different staining patterns: from left to right and top to bottom: double positive, single E-cadherin-positive, single NECTIN1-positive, double negative. Scale bar: 10 μm. BV, blood vessel. Representative examples of the cases described in g . ( g ) Distribution of NECTIN1 and E-cadherin positivity by immunofluorescence in 20 sections of human melanoma. Correlation was measured by Chi-square test.

Article Snippet: In addition, two commercial human melanoma tissue-microarrays were purchased from Biomax (ME1004e and ME2082c).

Techniques: Expressing, Whisker Assay, Two Tailed Test, Immunohistochemistry, Staining, Immunofluorescence

Journal: Journal of Cancer

Article Title: Suppression of CCT3 inhibits melanoma cell proliferation by downregulating CDK1 expression

doi: 10.7150/jca.69497

Figure Lengend Snippet: Global changes in melanoma cells transcriptome following the knockdown (KD) of CCT3 . (A) Hierarchical cluster analysis of differentially expressed genes with fold change of > 2.0. Row and column represent gene and experimental cells, respectively. Upregulated and downregulated genes are shown in red and green, respectively. (B) Signaling enrichment analysis of CCT3-downregulated classical signaling pathways based on IPA. Differential data are presented as z-scores (based on Fold change). (C) The expression trends of molecules in cyclins and cell cycle regulation pathway (based on IPA). Red and green represent upregulated and downregulated genes, respectively. (D) Knowledge-based interaction network of CCT3 targets after comparing the CCT3-KD and the shCtrl cells. The network was built on the basis of the CCT3 interactome of microarray data with a 1.5-fold change cutoff. The intensity of the node color indicates the degree of upregulation (red) or downregulation (green). Light colors represent less significant p values.

Article Snippet: A human melanoma tissue microarray (TMA-ME803a) that contains 40 patient samples was purchased from Genechem Co.,Ltd.

Techniques: Knockdown, Protein-Protein interactions, Expressing, Microarray