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. 2011 Apr 26;104(9):1459–1471. doi: 10.1038/bjc.2011.111

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Copyright © 2011 Cancer Research UK

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 https://creativecommons.org/licenses/by/4.0/.

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Bag-1 knockdown in the epidermal SCC cell line SCC-13 decreases cell yield and sensitises to 5-FU-induced apoptosis. Attached (A) and floating (B) cell counts from SCC-13 cells transfected with 10 nM of an irrelevant Silencer control siRNA, or Bag-1 small interfering RNA (siRNA) (a mixture of three Bag-1 siRNA sequences). Cells were treated 48 h after transfection with vehicle control (DMSO) or 20 μM 5-FU. Cell counts were performed at 48 h post treatment. Data shown are the mean values obtained from three independent experiments, each performed in triplicate. Error bars represent standard error of the mean. Statistical analysis of attached cell yields and percentage of cells floating indicated a highly significant (P<0.001) difference between Bag-1 siRNA and Silencer control siRNA under all treatment conditions (analysis of variance and Tukey's post hoc test). (C) The Bag-1 and Hsp70 levels in SCC-13 cells transfected with 10 nM of an irrelevant control siRNA, or Bag-1 siRNA (a mixture of three Bag-1 siRNA sequences). Untransfected cells treated with oligofectamine alone were included as a control to show that the Silencer control siRNA does not significantly affect basal levels of Bag-1 or Hsp70. Note that Hsp70 levels are unaltered by Bag-1 knockdown. (D) Cleavage of Poly (ADP-ribose) polymerase (PARP) induced by 5-FU is enhanced subsequent to Bag-1 knockdown. The Hsp70 levels are unaffected by Bag-1 knockdown or by 5-FU treatment. Similar results were obtained in a repeat experiment. (E) Acridine orange/ethidium bromide staining of Bag-1 siRNA transfected SCC-13 cells treated with 5-FU or vehicle control (DMSO). (a) Attached cells treated with vehicle control (DMSO). Some occasional spontaneous apoptotic floating cells were seen (inset). (b) Attached cells treated with 5-FU. Note the presence of some apoptotic cells amongst the attached cell population in Bag-1 siRNA-transfected SCC-13 (arrows). (c) Floating cells from 5-FU-treated SCC-13. The majority of cells show classical early apoptotic morphology (condensed chromatin, cell shrinkage, maintenance of membrane potential; i.e., cells do not take up ethidium bromide and hence appear green). Images a and b were photographed using a × 20 objective (scale bar shown in a). Image c was photographed using a × 10 objective (scale bar shown in c). The images have been cropped and enlarged to more clearly show the floating cells present.