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. 2021 May 2;12(5):438. doi: 10.1038/s41419-021-03707-7

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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 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. 8 — A, B ITIH5 was overexpressed in Mel-RM cells with or without KLF4 knockout (KO). Representative images of culture plates (A) and average numbers of colonies (B) from the colony formation assays. Data in B represent three independent experiments, ***p < 0.001, ns not statistically significant. Scale bar, 1 cm. C, D Represented images of crystal violet-stained culture plates (C) and average number of migrated cells (D) from Transwell cell migration assays. Data in D represent three independent experiments, ***p < 0.001. Scale bar, 100 μm. E, F ITIH5 was overexpressed in ME4405 cells. The protein and mRNA levels of NUCB2 were analysed by western blotting (E) and RT-qPCR (F). GAPDH was used as the loading control. Data in F represent three independent experiments, ***p < 0.001. G, H ITIH5 expression was knocked down in Mel-RM cells. The protein and mRNA levels of NUCB2 were analysed by western blotting (G) and RT-qPCR (H). GAPDH was used as the loading control. Data in H represent three independent experiments, **p < 0.01. I Schematic illustration of pGL3-based reporter constructs used to assess the transcriptional activity of NUCB2 by luciferase assay. The promoters of NUCB2, P1 and ITIH5 were cotransfected into Mel-RM cells with or without KLF4 knockout (KO). The luciferase activity of P1 was measured. The results represent three independent experiments, ***p < 0.001. J, K ChIP analysis showed the binding of KLF4 to the promoter of NUCB2 in Mel-RM cells with or without ITIH5 overexpression (J) or knockdown (K). An isotype-matched IgG was used as a negative control. L Schematic diagram depicting the upregulation of ITIH5 expression by p53 that suppresses melanoma growth and migration through the negative regulation of KLF4 transcriptional activity.