Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Identification of NRAS isoform 2 overexpression as a mechanism facilitating BRAF inhibitor resistance in malignant melanoma

doi: 10.1073/pnas.1704371114

Figure Lengend Snippet: NRAS isoform 2 is the aggressive isoform in melanoma. (A) Expression analysis by qRT-PCR of NRAS isoforms 1–5 mRNA in nine melanoma cell lines (circles) and human epidermal melanocytes (HEMs, squares; circled points, three BRAF mutant lines noted with asterisks in B). (B) NRAS isoform expression from same melanoma cell lines in A, visualized as expression levels relative to HEM. Bars represent three biological replicates normalized to 18S as a housekeeping gene, displayed as mean ± SEM. # indicates the presence of a BRAF mutation. (C) A375 cells that overexpress either NRAS isoform 1 or 2 or the A375 line transduced with empty vector were injected s.c. into nude mice (1 × 106 cells) and tumor growth was followed, displayed as mean ± SD. (D) Mice were removed from the study when tumors reached 800 mm3. Survival curves are depicted. (E) Tumor growth was also measured via fluorescent imaging of the GFP-expressing tumor cells at an exposure time of 1 s. Representative images of tumor fluorescence at day 14 are shown and radiant efficiency is quantified at day 8 and day 14, displayed as mean ± SD. Xenogen IVIS software was used to quantify fluorescence. (F) Whole tumors were harvested and sectioned to estimate levels of apoptosis and proliferation by immunohistochemical staining for cleaved caspase 3 and Ki67 staining, respectively. Representative images are displayed, as well as quantification of staining for n = 3 mice per group. For all in vivo figures, there are n = 5 mice per group. Significance was determined using an unpaired t test in all figures except for D where a log-rank test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Paraffin-embedded tissue was cut at 4 µm and stained with the following antibodies: Ki67 (AB16667; Abcam), cleaved caspase 3 (9,664; Cell Signaling).

Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Transduction, Plasmid Preparation, Injection, Imaging, Fluorescence, Software, Immunohistochemical staining, Staining, In Vivo