The effect of phosphorylation-mimetic mutants on the formation of cytoplasmic inclusions in cells. (a) p62 wild-type and mutants (S349E and S349A) fused with EGFP were transfected into HeLa cells. After 24 h, the cells were double-stained with rabbit anti-P-S349 (red) and mouse anti-pan-p62 (purple) antibodies. Wild-type p62 forms cytoplasmic inclusions in cells. The higher number of cytoplasmic inclusions is observed in cells transfected with the phosphorylation-mimetic mutant (S349E) compared with cells containing p62 wild-type and the S349A mutant (phosphorylation defective). An anti-phosphorylated p62 antibody reacts with S349E in inclusions, whereas this antibody only partially recognises inclusions in cells with p62 wild-type and S349A. Epo treatment increases the formation of S349 inclusions and p62 accumulation. The islet panels indicate higher magnification. Scale bar = 20 μm. (b) DEM also enhances the formation of p62 (blue) and phosphorylation of S349 (green). Keap1 (red) also colocalised with P-S349 signals. The islet panels indicate higher magnification. Scale bar = 20 μm. (c) Cells were treated with or without DEM for 24 h, and with transfection reagent alone (Fugene 6 or Lipofectamine 2000). Cells were harvested and analysed by immunoblotting. P-S349 signals are detected in samples treated with DEM (lane 2) and transfection reagents (lanes 4 and 5). (d) Knockdown of p62, Keap1 or Nrf2 was performed using Lipofectamine RNAi MAX. After 24 h, cells were treated with Epo for 24 h and harvested. Epo treatment enhances the P-S349 signal in cells treated with control siRNA compared to vehicle alone. No signals for P-S349 are observed in cells subjected to siRNA against p62 and siKeap1. Similar to other transfection reagents, Lipofectamine RNAi MAX slightly induced the phosphorylation of p62 at S349 (lane 1).