N-myc Downstream Regulated 1 (NDRG1) Is Regulated by Eukaryotic Initiation Factor 3a (eIF3a) during Cellular Stress Caused by Iron Depletion
Figure 1
The distribution of eIF3a and NDRG1 after incubation of MCF7 cells with DFO (250 µM), 311 (25 µM), tunicamycin (5 µg/mL) or hypoxia (1% O2).
(A) Cells were incubated using these conditions for 24 h/37°C and then stained with primary antibody against eIF3a (Alexa Fluor 555; “red”) and DAPI. Scale bar: 10 µm. (B) Co-localization of the stress granule markers eIF3a and eIF2α (Alexa Fluor 488; “green”) in structures consistent with stress granules after incubation with DFO under the conditions used in (A). The enlarged views of the boxed region in the merge panel are displayed to the right of this panel and show separate views for the red and green channels of the same field in which there is a cluster of eIF3a- and eIF2α-positive stress granules. The white arrows point to these structures. The scale bar in the left-most merge image represents 10 µm, while the scale bar in the enlarged merge image represents 2 µm. (C) Fractionation of MCF7 cells followed by western analysis demonstrated the presence of eIF3a and NDRG1 in both the cytoplasm and nucleus. GAPDH and HDAC1 were used as positive and loading controls for isolation of cytoplasmic (C) and nuclear (N) fractions, respectively. β-actin was used as general protein loading control. The blots are representative of 3 experiments and the densitometric analysis is expressed as mean ± SD. *p<0.05, **p<0.01, ***p<0.001 relative to the control of the same fraction.