Figure 1.
Different forms of oxidative stress target importin-α family members to SGs.
HeLa cells were exposed to DEM or arsenite. Importin-α1, α4 and α5 as well as the SG marker HuR were located by indirect immunofluorescence. Nuclei were stained with DAPI; size bar is 20 µm. Co-localization of HuR and importin-α family members is shown for the selected regions (white square in overlay panel) at a magnification of 500×.
Figure 2.
Oxidants DEM and arsenite promote the association of importin-α family members with SGs.
Experiments shown in Fig. 1 were performed with the SG marker G3BP1. Nuclei were detected with DAPI; size bar is 20 µm. Co-localization of G3BP1 and members of the importin-α family can be seen in the selected regions (white square in overlay panel) at a magnification of 500×.
Figure 3.
Heat shock induces the association of importin-α1, α4 and α5 with SGs.
Following a 1.5-hour heat shock, the distribution of importin-α proteins and SG marker HuR (A) or G3BP1 (B) was determined. DAPI was used to stain DNA; size bar is 20 µm. The area demarcated by the white square shows a 500× magnified view of SGs.
Figure 4.
Importin-β1, but not CAS, binds to SGs in response to oxidative stress.
HeLa cells treated with DEM or arsenite were stained with antibodies against the carrier importin-β1 or CAS. Co-staining was performed with the SG marker HuR; nuclei were detected with DAPI. Size bar is 20 µm; SG-containing areas (white squares) are magnified 500×.
Figure 5.
Oxidant-induced SGs contain importin-β1, but not CAS.
The selective association of importin-β1 with SGs under oxidative stress conditions was examined with the SG marker G3BP1. DAPI was used to identify nuclei; size bar is 20 µm; SG-containing regions (white squares) are shown at 500× magnification.
Figure 6.
Heat shock targets importin-β1, but not CAS, to SGs.
In heat-shocked HeLa cells, importin-β1, CAS and HuR (A) or G3BP1 (B) were detected by indirect immunofluorescence. DNA was stained with DAPI; size bar is 20 µm. Magnified views (500×) of SG-containing regions are depicted for stressed cells.
Figure 7.
Quantification of the SG association for importin-α1, α4, α5 and importin-β1.
HeLa cells were incubated with DEM, arsenite or heat shock and stained with antibodies against importin-α1, α4, α5, or importin-β1. SGs were identified with the marker protein HuR, and 30 SG-containing cells were evaluated for every treatment. Each individual SG was scored for the presence of importin-α1, α4, α5 or importin-β1. A single data point represents the average of three independent experiments+SEM. The tables depict the number of SGs that were positive for the importin analyzed/number of SGs identified with HuR; results are shown for each individual experiment. Note that more than 90% of SGs were positive for the examined members of the importin-α family or importin-β1.
Figure 8.
Transport factors importin-α1, α4, α5, importin-β1 and CAS do not associate with PBs under normal or stress conditions.
Transport factors and the PB marker Dcp1 were detected in HeLa cells treated with DEM as described for Fig. 1. Nuclei were stained with DAPI; size bar is 20 µm. Selected PB-containing regions are shown at a magnification of 500×.
Figure 9.
The association of importin-α1 with poly(A)-RNA in growing cells is regulated by stress.
Crude extracts prepared for controls (ethanol, EtOH) and DEM-treated cells were incubated with oligo-(dT)-cellulose as described in Materials and methods. Aliquots of the start (St, 10%) and eluted (E, 100%) material were analyzed by Western blotting with different antibodies as indicated. The relative binding was calculated for importin-α1 and HuR; results were normalized to control samples. Data for three independent experiments are shown as average +SEM. Student’s t-test was applied to identify significant differences between control and DEM-treated samples; *, p<0.05. There was no significant change for HuR.
Figure 10.
Interaction of purified importin-α1 with RNA homopolymers in vitro.
His6-importin-α1 or His6-poly(A)-binding protein was synthesized in E. coli. Purified importin-α1 was incubated with immobilized poly(A), poly(U), poly(C) or poly(G) homopolymers or non-conjugated resin. In control experiments, resins were pre-treated with micrococcal nuclease as indicated. Aliquots of the flow through (10%, Ft) and bound material (100%, B) were analyzed by Western blotting with antibodies against importin-α1 (top). Under the same conditions, a strong interaction between immobilized poly(A) and purified poly(A)-binding protein was detected with antibodies against the His-tag (bottom).
Table 1.
Summary of results for the association of nuclear transport factors with SGs and PBs.