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 3

Levels of p62 mRNA, its binding by HuR protein, and de novo translation following AICAR + MG132 exposure. (a) Representative Western blotting (upper) and densitometric analyses (lower) of p62 protein levels in the total homogenates of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for 2 hrs in the presence or not of puromycin (1 μM, PURO). Optical densities of p62 bands were normalized to α-tubulin, and the results expressed as mean percentages + S.E.M. (; and ; Tukey’s multiple comparisons test). (b) Determination by real-time qPCR of p62 mRNA levels in the total homogenate (upper) and cytoplasm (lower) of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for 2 hrs. p62 mRNA levels were normalized in accordance with the corresponding RPL6 mRNA content. The values are expressed as mean percentages + S.E.M. The experiments were performed in duplicate on 4-5 independent sets of cells (, Student’s t test). (c) Fold enrichment detected by real-time qPCR of p62 mRNA following immunoprecipitation (IP) with either anti-HuR antibody or irrelevant antibody (Irr) in the cytoplasm of ARPE-19 cells exposed to either solvent (CTR) or AICAR + MG132 (A + M) for 2 hrs (; ; Tukey’s multiple comparison test). (d) Polysome profile of p62 mRNA was determined using 15–50% sucrose gradient sedimentation. (e) Real-time qPCR analysis and transcript level quantification for p62 were performed in free RNA (pooled fractions 3 and 4), monosomes (pooled fractions 5 and 6), and polysome (pooled fractions 7, 8, and 9) of ARPE-19 cells treated with either solvent (DMSO) or AICAR + MG132 for 2 hrs. Relative expression of p62 was normalized to mRNA of free RNA sample, considering the value of GAPDH as a housekeeping gene.

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