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. 2018 Nov 21;7(11):89. doi: 10.1038/s41389-018-0100-4

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — Stable eRMS (Rh18) and aRMS (Rh30) cells were pre-treated and maintained with 30 µg/mL cumate to induce PANX1 expression and 3D spheroid formation was monitored over 210 h. Representative images taken at 48 and 210 h clearly show that over-expression of PANX1 prevented eRMS (Rh18) (a) and aRMS (Rh30) (b) spheroid formation. Mean image fluorescence (MIF), a surrogate measurement for spheroid size, was measured over time for both eRMS (Rh18) (c) and aRMS (Rh30) (d) cells. A.U. arbitrary units. Bars = 1000 µm. The viability of stable eRMS (Rh18) (e; left panel) and aRMS (Rh30) (f; left panel) cells grown in suspension on agar for 10 days in the absence or presence of cumate was assessed every 2 days. Viable cell counts showed a significantly reduced number of viable cells within cumate-induced PANX1 RMS spheroids. ***P < 0.001 compared to untreated GFP cells, GFP cells treated with cumate, and untreated PANX1 cells. After being grown in suspension for 4 days, live (Annexin V−, 7-AAD−), early apoptotic (Annexin V+, 7-AAD-), late apoptotic (Annexin V+, 7-AAD+) and necrotic (Annexin V−, 7-AAD+) cell populations were quantified as percentages of the total cell population and revealed a significant increase in apoptotic cell populations within PANX1 over-expressing eRMS (Rh18) (e; right panel) and aRMS (Rh30) (f; right panel) spheroids. ***P < 0.001, **P < 0.01 compared to respective control cells expressing GFP. Results are expressed as mean ± s.d. In the spheroid regression assay, stable eRMS (Rh18) and aRMS (Rh30) cells were first allowed to form spheroids for 48 h before being treated with cumate to induce PANX1 expression. Spheroid growth was monitored for a total of 210 h. While the mean image fluorescence (MIF) of control stable eRMS (Rh18) (g) and aRMS (Rh30) (h) spheroids progressively increased over time, a continuous and significant decrease of MIF was measured upon PANX1 expression. ***P < 0.001 compared to untreated GFP cells, GFP cells treated with cumate, and untreated PANX1 cells. Results are expressed as mean ± s.d. A.U. arbitrary units. Bars = 1000 µm