Journal: Cancers

Article Title: Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth

doi: 10.3390/cancers14246247

Figure Lengend Snippet: NT157 treatment reduces IRS-1 levels leading to a reduction of cell viability, migration, and induction of apoptosis in UM cell lines. ( A ) TCGA database analysis shows high expression of IRS-1 mRNA in UM tumors (indicated by an arrow) among 33 cancer (red dots) and corresponding normal (green dots) tissues. ( B ) Immunohistochemical staining of matched primary (eye) and metastatic (liver) UM tumor tissues using IRS-1 antibody detects IRS-1 expression (purple) in both eye and liver (both 2× and 20× magnifications are shown). ( C ) Western blot analysis of cell extracts from UM cell lines (92.1, OMM2.5, OMM1, MEL20-06-039, MP65) treated with NT157 (1 µM) and probed with anti-IRS-1 antibody shows IRS-1 protein levels decrease in UM cell lines with NT157 treatment for 48 h. ( D ) Quantification of percent cell survival using MTT-based assays in UM cell lines (MP46, MM28, MEL202, 92.1, MP65, and MP41) with (0.05, 0.1, 0.25, 0.5, 1.0, 2.5 µM) or without NT157 treatment (72 h) shows NT157 dose-dependent decrease in cell survival. The bar graphs represent a percentage of cell survival and are a mean ± SD of three independent experiments. p-values were calculated by comparing untreated controls with NT157 treatment doses ( * p < 0.05; ** p < 0.01; *** p < 0.001). ( E ) Colony formation assay: Representative images of crystal violet staining shows fewer colonies formed by UM cell lines (MP46, MM28, MEL202, 92.1, MP65, and MP41) with NT157 treatment (1 and 2.5 µM for seven days) compared to untreated controls. ( F ) Representative FACS histograms of DNA content detected by propidium iodide staining to detect cell cycle status in UM cell lines (MP46, MM28 and MEL20-06-039, and MP65) show a dose-dependent increase in the number of cells in G0/G1 phase with NT157 treatment (1 and 2.5 µM for 72 h) vs. no treatment. ( G ) Western blot analysis of total cell extracts shows NT157 dose-dependent (1 and 2.5 µM for 72 h) increase in cleaved PARP and decrease in caspase 3 levels detected by respective antibodies in UM cell lines (MEL202, MEL270, 92.1, and MEL20-06-039) compared to untreated controls. ( H ) Quantification of cell migration in UM cell lines (OMM1, 92.1, MEL202, MP65, and MM28) shows a reduction in migration of cells treated with NT157 (2.5 µM) compared to untreated controls. An average of five fields of cells/filter were counted under a microscope with 40× magnification. The average number of cells counted/field were obtained in two independent experiments and the mean ± SD of these average cell counts/field were plotted as bar graphs. p -values were calculated by comparing untreated vs. NT157 treated cells ( * p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: UM cell lines obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) were: MM28 (#CRL-3295), MP38 (#CRL-3296), MP41 (#CRL-3297), MP46 (#CRL-3298), MP65 (#CRL-3299).

Techniques: Migration, Expressing, Immunohistochemical staining, Staining, Western Blot, Colony Assay, Microscopy

Journal: Cancers

Article Title: Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth

doi: 10.3390/cancers14246247

Figure Lengend Snippet: Shows percent cells gated in subG1 phase with /without treatment with NT157 . The percent of cells in cell cycle phases was obtained by counting propidium iodide-stained cells after 48 h of NT157 treatment. At the higher NT157 concentration we see an accumulation of subG1 cells compared to the untreated controls.

Article Snippet: UM cell lines obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) were: MM28 (#CRL-3295), MP38 (#CRL-3296), MP41 (#CRL-3297), MP46 (#CRL-3298), MP65 (#CRL-3299).

Techniques: Concentration Assay