Figure 6. Modulation of nucleo-cytoplasmic HuR shuttling and binding to the 5ʹUTR-caspase-2 by chemotherapeutic drugs.

(A) Doxorubicin-induced nuclear export of HuR in human colon carcinoma cells by indirect immunofluorescence. Serum-starved DLD-1 cells were treated for 6 h with either vehicle, or with 10 μg/ml doxorubicin (Doxo.) before cells were fixed and successively stained with anti-HuR and anti-mouse Alexa-488 antibodies. Thereafter, DAPI was added to counterstain cell nuclei (blue panel). bar: 50 μm. (B) Time-dependent increase in cytoplasmic HuR content by chemotherapeutic drugs. Serum-starved DLD-1 cells were treated with doxorubicin (Doxo.) or paclitaxel (Pacli.) as indicated, before cells were lyzed for cytoplasmic cell lysates and cytoplasmic (cytopl.) or nuclear (nucl.) HuR levels were subsequently monitored by Western blot analysis. Loading of equal amounts of cytoplasmic extracts was ascertained by reprobing the blots with anti-β-actin antibody and Lamin-B1 was used as a nuclear marker protein. Values at the bottom represent means ± SD (n = 3) ^*P ≤ 0.05, ^**P ≤ 0.01 compared with the corresponding vehicles. (C) Cells were serum starved for 16 h before treated either 6 h with doxorubicin (left panel), or 24 h with paclitaxel (right panel) in the absence (vehicle) or presence of either Gö6983 (100 nM), the Chk2 inhibitor II (Chk2 inhibit. 2 μM), KU55933 (10 μM), SB203580 (10 μM) and U0126 (20 μM) as indicated. All inhibitors were preincubated for 30 min prior to the addition of the chemotherapeutic drug. Values in the graphs represent means ± SD (n = 3) ^*P ≤ 0.05, ^**P ≤ 0.01 compared with the corresponding vehicles and ^#P ≤ 0.05, ^##P ≤ 0.01 compared with doxorubicin (left panel) or paclitaxel (right panel)-induced conditions.