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. 2017 Jun 30;32(1):111–119. doi: 10.1038/leu.2017.182

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Copyright © 2018 The Author(s)

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/4.0/

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Specific knockdown of NER-related genes increases sensitivity to melphalan in MMCLs. (a and b) ERCC3 and XPC knockdowns impair NER and increase sensitivity to melphalan. The figure represents the impact of XPC and ERCC3 knockdown on NER (left graph) and on melphalan sensitivity (right graph) in RPMI8226 and LR5 cell lines. Three independent experiments were processed 72 h after transfection with scrambled siRNA or siRNA targeting ERCC3 or XPC. The impact of knockdown on NER was evaluated with the measurement of remaining (6-4) photoproducts signal 120 min after UV-C exposure, using anti-(6-4) photoproducts antibody. Cell viability was evaluated by CellTiter-Glo (Promega Corporation, Madison, WI, USA). (c and d) Knockdown efficiency. Western blot evaluation of XPB and XPC levels 72 h after transfection with scrambled or specific siRNA. (e and f) Stable knockdown of ERCC3 increases sensitivity to melphalan. The figure represents the impact of ERCC3-stable knockdown in MM1S cell line on melphalan sensitivity. Cell viability was assessed by CellTiter-Glo 1 month after knockdown and puromycin selection. Confirmation of XPB knockdown was assessed by western blot. (g–i) Stable overexpression of ERCC3 increases resistance to melphalan. Apoptotic and cell death were assessed in MM1S and RPMI8226 cells transduced with either control (RFP-GFP-positive cells) or ERCC3-GFP particles (GFP-positive cells) by flow cytometric analysis following Annexin-V and propidium iodide (PI) staining after exposure to melphalan at several doses. The percentage of live cells corresponds to the proportion of lives cells on the total of GFP- or RFP-positive cells. Confirmation of XPB overexpression was assessed by western blot.