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. 2014 Jan 29;15(2):2172–2190. doi: 10.3390/ijms15022172

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© 2014 by the authors; licensee MDPI, Basel, Switzerland

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

PMC Copyright notice

Figure 5. — Int6 gene silencing induces cell apoptosis. (A) Dot plot representing the percentage of Annexin V positive cells in two GBM cell lines (LN18 and U87) analyzed by flow cytometry and Annexin V/Propidium Iodide staining; (B) Quantification of Annexin V positive cells. Int6 inhibition (siInt6) results in a significant increase in apoptotic cells (** p < 0.01, *** p < 0.001 compared with the negative control, siScr, n = 5); (C) Western Blot analysis and quantifications of caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, Bcl-2, Bcl-XL and Bax expression in LN18, SF767, U87 and U251 GBM cells transfected with siInt6 or siScr. Int6 silencing slightly increases Bax expression, reduces Bcl-2, caspase 3 and caspase 7 expression, and increases Bax/Bcl-2 ratio and cleaved caspase forms for U251 and LN18 cells (* p < 0.05, ** p < 0.01, n = 3); (D) Photographs representing glioma cells transfected with siScr or siInt6 and treated with 30 μM of Z-VAD caspase inhibitor (+Z-VAD) or DMSO (−Z-VAD). Z-VAD treatment partly rescues the deleterious effect of Int6 silencing (n = 3), scale bars: 20 μm; (E) Quantification of Annexin-V/PI staining analyzed by flow cytometry of glioma cells transfected with siScr or siInt6 and treated or not with Z-VAD caspase inhibitor. Z-VAD treatment partly reverses glioma cell death induced by Int6 inhibition (* p < 0.05, n = 3).

Figure 5. — Int6 gene silencing induces cell apoptosis. (A) Dot plot representing the percentage of Annexin V positive cells in two GBM cell lines (LN18 and U87) analyzed by flow cytometry and Annexin V/Propidium Iodide staining; (B) Quantification of Annexin V positive cells. Int6 inhibition (siInt6) results in a significant increase in apoptotic cells (** p < 0.01, *** p < 0.001 compared with the negative control, siScr, n = 5); (C) Western Blot analysis and quantifications of caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, Bcl-2, Bcl-XL and Bax expression in LN18, SF767, U87 and U251 GBM cells transfected with siInt6 or siScr. Int6 silencing slightly increases Bax expression, reduces Bcl-2, caspase 3 and caspase 7 expression, and increases Bax/Bcl-2 ratio and cleaved caspase forms for U251 and LN18 cells (* p < 0.05, ** p < 0.01, n = 3); (D) Photographs representing glioma cells transfected with siScr or siInt6 and treated with 30 μM of Z-VAD caspase inhibitor (+Z-VAD) or DMSO (−Z-VAD). Z-VAD treatment partly rescues the deleterious effect of Int6 silencing (n = 3), scale bars: 20 μm; (E) Quantification of Annexin-V/PI staining analyzed by flow cytometry of glioma cells transfected with siScr or siInt6 and treated or not with Z-VAD caspase inhibitor. Z-VAD treatment partly reverses glioma cell death induced by Int6 inhibition (* p < 0.05, n = 3).

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