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. 2016 Jun 27;5(6):e236. doi: 10.1038/oncsis.2016.45

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Copyright © 2016 Macmillan Publishers Limited

Oncogenesis is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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GNAQ^Q209L-driven secondary melanoma tumor progression is attenuated by induced Ric-8A deletion. (a) Two explanted GNAQ^Q209L tumors from Figure 3a were cultured ex vivo in standard melanocyte culture medium lacking TPA to establish Ric-8A^Flox/Flox; Rosa-CreER^+/⁻; (Tg) GNAQ^Q209L melanoma cell lines. The growth kinetics of the melanoma cell lines±TPA supplementation and ±4OHT to induce ex vivo Ric-8A deletion as indicated were compared with the growth progression of the original Ric-8A^Flox/Flox; Rosa-CreER^+/⁻; (Tg) GNAQ^Q209L melanocyte cell line. (b) Bright-field images of the parental Ric-8A^Flox/Flox; Rosa-CreER^+/⁻; (Tg) GNAQ^Q209L melanocyte cell line and melanoma explant culture #2 at the conclusion of the 6-day growth study. Error bars are the mean±s.e.m. of experiments performed in triplicate. (c) Quantitative western blot analyses of Ric-8A, Gαq/11, Gαq-Q209L and Gα13 levels in lysates prepared from the parental Ric-8A^Flox/Flox; Rosa-CreER^+/−; (Tg) GNAQ^Q209L melanocyte cell line and melanoma cell cultures derived from the two independent explanted GNAQ^Q209L-melanoma tumors. Tumor cell cultures were treated with 4OHT to induce Ric-8A knockout ex vivo. α-Tubulin levels were measured as a normalization control. (d) Secondary tumor progression from grafted Ric-8A^Flox/Flox; Rosa-CreER^+/⁻; (Tg) GNAQ^Q209L explant cultures #1 and #2 after in vitro treatment with solvent or 4OHT to induce Ric-8A deletion. Shown alongside are images and average weights of harvested secondary tumors at day 56 (culture #2) and day 71 (culture #1). Error bars are the mean±s.e.m. (n=4 tumors).