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. Author manuscript; available in PMC: 2011 Mar 16.

Published in final edited form as: Cancer Biol Ther. 2009 Jan;8(1):31–35. doi: 10.4161/cbt.8.1.7079

COX-2-silenced cells accumulate functional HIF-1α and maintain an intact hypoxic response. (A) Immunoblotting for HIF-1α in COX-2-containing parental MDAMB-231 (231) cells, and COX-2-silenced Clone 2 and Pooled cells subjected to the indicated amounts of CoCl₂ for 24 h. Total cell lysate from each cell line was subjected to immunoblotting for HIF-1α, COX-2 and GAPDH. (B) Immunoblotting for HIF-1α in COX-2-containing parental MDA-MB-231 (231) cells, and COX-2-silenced Clone 2 and Pooled cells subjected to 0.7% hypoxia for the indicated times. Total cell lysate from each cell line was subjected to immunoblotting for HIF-1α, COX-2 and GAPDH. (C) Fold induction of HIF-1α-induced transcripts SLC2A1, VEGF and LDHA in COX-2-containing (MDA-MB-231) and COX-2-silenced cells (Clone 2 and Pooled) in response to 24 h of hypoxia. Fold induction was calculated using the 2^ΔΔCt method using 18S Ct values as control. (D) Microarray analysis of total mRNA in COX-2-containing (MDA-MB-231) and COX-2-silenced cells (Clone 2 and Pooled) in response to hypoxia for 24 h. Fold-induction of known HIF-1α target genes is shown. (E) Fold induction of transcripts significantly upregulated by hypoxia in MDA-MB-231 cells and comparison with the fold induction of transcripts that were similarly regulated between Clone 2 and Pooled cells.

COX-2-silenced cells accumulate functional HIF-1α and maintain an intact hypoxic response. (A) Immunoblotting for HIF-1α in COX-2-containing parental MDAMB-231 (231) cells, and COX-2-silenced Clone 2 and Pooled cells subjected to the indicated amounts of CoCl₂ for 24 h. Total cell lysate from each cell line was subjected to immunoblotting for HIF-1α, COX-2 and GAPDH. (B) Immunoblotting for HIF-1α in COX-2-containing parental MDA-MB-231 (231) cells, and COX-2-silenced Clone 2 and Pooled cells subjected to 0.7% hypoxia for the indicated times. Total cell lysate from each cell line was subjected to immunoblotting for HIF-1α, COX-2 and GAPDH. (C) Fold induction of HIF-1α-induced transcripts SLC2A1, VEGF and LDHA in COX-2-containing (MDA-MB-231) and COX-2-silenced cells (Clone 2 and Pooled) in response to 24 h of hypoxia. Fold induction was calculated using the 2^ΔΔCt method using 18S Ct values as control. (D) Microarray analysis of total mRNA in COX-2-containing (MDA-MB-231) and COX-2-silenced cells (Clone 2 and Pooled) in response to hypoxia for 24 h. Fold-induction of known HIF-1α target genes is shown. (E) Fold induction of transcripts significantly upregulated by hypoxia in MDA-MB-231 cells and comparison with the fold induction of transcripts that were similarly regulated between Clone 2 and Pooled cells.