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. 2018 Feb 15;8:3081. doi: 10.1038/s41598-018-21361-6

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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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Knockdown of SDHB in HCC Hep3B cells greatly increases tumor cell proliferation and migration. (A) Cell morphology of SDHB-knockdown cells. Cells were grown for 48 h and imaged microscopically. (B) Western blot analysis of SDHB protein in SDHB-knockdown cells. Total proteins isolated from cells as indicated were blotted with antibodies against SDHB and β-actin. (C) SDH activity assay in SDHB-knockdown cells. Cells of equal-numbers, as indicated, were subjected to a MTT assay according to standard protocols. (D) SDHB-knockdown cell counts. Cells as indicated were cultured in 6-well plates, and 10 random fields were imaged microscopically. Cells in the 10 fields were scored. (E) Colony formation assay of SDHB-knockdown cells. Cells as indicated were seeded into 6-well plates for 7 days. The colonies obtained were stained and scored. (F) Wound healing migration assay of SDHB-knockdown cells. Cells as indicated were grown until confluent before a scratch wound healing migration assay was performed. (G) Boyden chamber migration assay of SDHB-knockdown cells. Cells as indicated were plated in a Boyden chamber and incubated for 8 h. The migrated cells were stained and counted. (H) In vivo tumor growth assay of SDHB-knockdown cells. Cells as indicated were subcutaneously inoculated into the back of NOD/SCID mice for 14 days. The mice were sacrificed and the tumors removed and examined. (I) Tumor volumes were measured after removal. SDHB-knockdown cells exhibited a larger tumor volume compared with mock and vector-transfected cells. (J) Histological and immunohistochemical staining of the lung and heart in the in vivo tumor metastasis assay. Cells as indicated were intravenously injected into the tail vein of NOD/SCID mice for 20 days. Mice were sacrificed and examined for tumor metastasis. Experiments were carried out using H&E staining and an antibody specific for vimentin. The β-actin level serves as a control for protein loading. The *^, ** and *** represented P-value < 0.05, <0.01 and <0.005, respectively.