Autophagy Stimulus Promotes Early HuR Protein Activation and p62/SQSTM1 Protein Synthesis in ARPE-19 Cells by Triggering Erk1/2, p38^MAPK, and JNK Kinase Pathways

Figure 1

Translocation of HuR protein following AICAR + MG132 exposure. (a) Representative Western blotting (upper) and densitometric analysis (lower) of HuR protein levels in the nucleus (a), cytoplasm (b), and total homogenate (c) of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for increasing times (15, 30, and 120 min). Optical densities of HuR bands were normalized to lamin C for the nucleus and to α-tubulin for both the cytoplasm and total homogenate. The same proteins were also used as purity controls for each cellular fraction. The values are expressed as mean percentages + S.E.M. (; and ; Dunnett’s multiple comparison test). (d, e) Representative Western blotting (upper) and densitometric analyses (lower) of HuR protein phosphorylated in threonine residues (p-Thr HuR; (d)) and serine residues (p-Ser HuR; (e)) in the cytoplasm of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for increasing times (15, 30, and 120 min). Optical densities of phosphorylated HuR bands were normalized to α-tubulin (loading control detected in the input signals), and the results expressed as mean percentages + S.E.M. (; ; Dunnett’s multiple comparison test). (f) Representative immunocytochemistry images of HuR protein in ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 for 2 hrs. The left panels show HuR staining (red), the right panels show nuclei staining with DAPI (blue), and the middle panels merged images. Imaging (40x magnification) was made with a PerkinElmer image plate reader Operetta. Scale bar: 20 μm. Inserts: immunofluorescence analysis of HuR was performed using a Zeiss Observer Z1 microscope equipped with Apotome module, with a Plan Apochromatic (63x, NA 1.4) objective. Nuclei staining with DAPI (blue). Images were acquired using Zen 1.1 (blue edition) imaging software and assembled with ImageJ software.

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