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. 2020 May 15;9(5):51. doi: 10.1038/s41389-020-0231-2

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© The Author(s) 2020

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. 2 — a–c Proliferation curves of three stable cell lines (SW1990 (a), PANC-1 (b), PaTu-8988t (c)) with knockdown (KD) of COX6B2 compared with corresponding control (Ctrl) cells. d Colony formation assays of COX6B2 KD PANC-1 (upper panel) and COX6B2 KD PaTu-8988t (middle panel) cells, and cells with re-expression of COX6B2 (lower panel) compared with corresponding control cells. Histograms on the right show the quantitative results, respectively. e Tumor xenograft experiments of PANC-1 and PaTu-8988t cells (5 × 10⁶ cells/mouse). All mice were sacrificed after 9 (PANC-1) or 8 (PaTu-8988t) weeks to measure the final tumor weight. (n = 6 mice per group). f–i Wound healing assays of 3 stable cell lines (SW1990 (f), PANC-1 (g), PaTu-8988t (h)) with knockdown of COX6B2, and of a COX6B2 KD PaTu-8988t cell line with re-expression of COX6B2 (i) compared with corresponding control cells. j–m Transwell assays show the migration (upper panel) and invasion (lower panel) abilities of three stable cell lines (SW1990 (j), PANC-1 (k), PaTu-8988t (l) with knockdown of COX6B2, and cell line with re-expression of COX6B2 (m). n Transwell assays of the HPNE (hTERT-HPNE) normal pancreatic ductal epithelial cell line and five PDAC cell lines as indicated. o Representative immunofluorescence photomicrographs of COX6B2 KD PaTu-8988t cells compared with control cells. Photomicrographs were captured using a confocal laser microscope (600× magnification, scale bar = 25 μm). Actin cytoskeleton and nuclei were stained with anti-F-actin (green) and DAPI (blue), respectively. p, q Representative images of macro-anatomy (left panel), lung (Lu) (middle upper panel), liver (L) (middle lower panel), and paired H&E-stained images after tail vein injection with control PANC-1 cells (p) and PANC-1 cells with KD of COX6B2 (q). For 40× magnification, scale bar = 50 μm; For 100× magnification, scale bar = 20 μm. Arrows indicate metastatic sites in abdominal cavity: gut (G), mesentery (M), pancreatic (P), lymph nodes (Ln), and pleura (Pl). T tumor, N adjacent normal tissue. n = 5 mice per group. r Average number of metastatic tumor nodules in mouse after tail vein injection with control PANC-1 cells and PANC-1 cells with KD of COX6B2. n = 5 mice per group. Histograms show the quantitative results respectively. All data are presented as mean ± SEM (n ≥ 3). *P < 0.05, **P < 0.01 and ***P < 0.001. NS no significance.