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Figure 7 | Cancer Cell International

Figure 7

From: Targeted inhibition of heat shock protein 90 disrupts multiple oncogenic signaling pathways, thus inducing cell cycle arrest and programmed cell death in human urinary bladder cancer cell lines

Figure 7

NF-κΒ inactivation after exposure of human bladder cancer cells to geldanamycin. NF-κB transcriptional capacity is diminished upon 24-hour geldanamycin administration in RT4 and T24 bladder cancer cells. (A) Upper panel: Western blottings displaying NF-κB protein expression levels and compartmentalization responses to drug treatment in both cytoplasmic and nuclear protein extracts of RT4 and T24 cells. Protein sample quantification of cytoplasmic and nuclear extracts was evaluated using Actin and Lamin A/C as respective (compartment-specific) reference markers, while the absence of detectable NF-κB in nuclear extracts derived from drug-treated cells ensured samples’ purity. Lower panel: cytoplasmic and nuclear NF-κB densitometric quantification bars, denoting its drug-induced compartmentalization change compared to control conditions, using Actin and Lamin A/C as respective proteins of reference. Standard deviation values are depicted as error bars on top of each value. Experiments were carried out three times, while characteristic Western blotting results are presented here. (B) Electrophoretic mobility shift assay (EMSA) conducted on RT4 and T24 nuclear protein extracts. Binding assays were performed using NF-κB-specific and 5´ -biotin labeled, double-stranded (annealed), oligonucleotides. EMSA experiments were repeated three times, one of which is shown here. (C) Left panel: transcriptional expression profiles of four critical anti-apoptotic NF-κB target genes (Survivin, cIAP-1, cIAP-2 and XIAP), in response to 24-hour geldanamycin administration, in RT4 and T24 bladder cancer cells. Right panel: RNA transcript densitometric quantification bars, denoting the drug-induced expression level alterations of the NF-κB target genes examined, compared to control conditions, using GAPDH (left panel) as gene of reference. All sqRT-PCR reactions were carried out three times, while a characteristic assembled profiling is presented here. Standard deviation values are depicted as error bars on top of each value.

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