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. 2021 Sep 15;11:18405. doi: 10.1038/s41598-021-97791-6

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Overexpression of PITX1 inhibits melanoma proliferation. (A) A2058 and SKMEL28 cell lines were infected with control and PITX1 expression lentivirus vectors. Infection efficiency was monitored after 48 h or 96 h by fluorescence analysis of vector-encoded GFP (right panels). Phase contrast images are shown at left. Scale bars: 100 µm. (B) Number of PITX1 transfected cells and control cells over 5 days. A2058 cells are shown at left, and SKMEL28 cells are shown at right. Bars correspond to means ± S.D. for three independent experiments (P < 0.001). Curve images of growth were created using Excel. (C,D) Flow cytometric analysis of apoptosis in PITX1 overexpressing cells (C: A2058, D: SKMEL28). Quantification of apoptosis as % Annexin V positive cells in GFP positive gate. Bars correspond to means ± S.D. of three independent experiments (P < 0.001). Annexin V-positive cell count images were created using Kaluza software, and the bar graphs were created using Excel. (E,F) ATP assay in PITX1-overexpressing cells (E: A2058, F: SKMEL28). ATP content was measured using CellTiter-Glo 2.0 After Assay Kit at 96 h after infection with PITX1 expressing or control lentivirus vector. Values are expressed as a percentage of control. Bars correspond to the means ± SD of three independent experiments (P < 0.001). These bar graphs were created using Excel.