Transdifferentiation of Melanoma Cells by the Reprogramming Factors Attenuates Malignant Nature In Vitro and In Vivo  Mikiro Takaishi, Shigetoshi Sano  Journal of Investigative Dermatology  Volume 139, Issue 1, Pages 254-257 (January 2019) DOI: 10.1016/j.jid.2018.06.179 Copyright © 2018 The Authors Terms and Conditions

Figure 1 Introduction of the reprogramming factors into A2058-induced transdifferentiation. (a) Morphology of parental A2058 cells and RICs in monolayer culture, phase contrast view (top panels). F-actin (middle panels) and MITF in nuclei (bottom panels) were observed with a fluorescence microscope. Scale bars = 100 μm in top panels and 50 μm in middle and bottom panels. (b) Relative mRNAs in the parental cells and RICs were studied by quantitative reverse transcription PCR, normalized with a value of hypoxanthine phosphoribosyl transferase (HPRT). n = 3, mean ± standard deviation. ∗∗P < 0.01, unpaired t test. (c) Western blot analysis. P indicates parental cells and R indicates RICs. β-Actin (ACTB) and HPRT were used as loading controls. NS, not significant; RIC, reprogramming factor-introduced cancer cell. Journal of Investigative Dermatology 2019 139, 254-257DOI: (10.1016/j.jid.2018.06.179) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Melanoma RICs showed attenuated malignancy in vitro and in vivo. (a) Cell proliferation in vitro was determined by number counting over time. No significant difference was observed between the parental cells and RICs. n = 3, mean ± standard deviation. Unpaired t test. (b) Cell migration activity was determined by in vitro wounding assay. The distance of cell migration from the wound edge was measured at 12 hours. n = 3, mean ± standard deviation. ∗∗P < 0.01, unpaired t test. (c) In vitro invasion assay was performed using Boyden chambers coated with Matrigel (BD, Bedford, MA). Migrated cells through the membrane of the lower chamber were counted at 24 hours after inoculation. n = 3, mean ± standard deviation. ∗∗P < 0.01, unpaired t test. (d) The survival rate of SCID mice inoculated with the RICs or parental A2058 cells. 1 × 106 cells/mouse, n = 10. Combined data from three independent experiments are shown. Log-rank test. (e) Left panel, multiple tumor foci in the lung and mediastinum of a mouse transplanted with A2058 parental cells at day 36. Right panel, lung of a mouse transplanted with RICs at day 90. Arrows indicate tumor foci. (f) Histology of the tumors in the lungs. Top panels, hematoxylin and eosin. Middle panels, immunostaining for MITF. Bottom panels, immunostaining for Ki67. Scale bars = 500 μm in top and middle panels and 50 μm in bottom panels. (g) Number of Ki67-positive cells in the tumors in five non-overlapping fields. Mean ± standard deviation. ∗∗P < 0.01, unpaired t test. D, day; RIC, reprogramming factor-introduced cancer cell. Journal of Investigative Dermatology 2019 139, 254-257DOI: (10.1016/j.jid.2018.06.179) Copyright © 2018 The Authors Terms and Conditions