FIG 5.

PPY-based iron chelators induce expression of IκB-α and affect NF-κB cellular distribution. (A) 293T cells were treated with 10 μM PPY, PPYaT, or PPYeT. DMSO was used as a vehicle control. After 24 h of treatment, RNAs were extracted, reverse transcribed, and analyzed by real-time PCR for CDK2, cyclin A, cyclin E, HLA, and IκB-α, using 18S RNA as a housekeeping control gene. (B) 293T cells were treated as described for panel A and then lysed in SDS-PAGE loading buffer, resolved by 10% SDS-PAGE, and probed with antibodies against IκB-α, phosphorylated IκB-α, and tubulin, as a loading control. Results were quantified using ImageQuant software. Data are representative of two independent experiments. In the bottom panel, averages of results from two independent experiments are shown. (C) 293T cells were treated as described for panel A, fixed, and stained with primary antibodies against the NF-κB p65 subunit and with fluorescein isothiocyanate (FITC)-linked secondary antibodies. Photographs were taken on an Olympus IX 51 microscope at a magnification of ×200, and the pictures were scored for the distribution of NF-κB localized only in the cytoplasm or both in the nucleus and in the cytoplasm. Average data obtained from 6 separate fields are shown. (D) NF-κB p65 expression was analyzed by resolving cytoplasmic and nuclear extracts by 10% SDS-PAGE and immunoblotting with antibodies against the NF-κB p65 subunit and tubulin, as a loading control.