Knock down of SPBP impairs p62 expression and sulforaphane induced p62 body formation.

<p>(<b>A</b>) Endogenous SPBP and p62 are coexpressed in several cell lines. Extracts of the indicated human cell lines were analysed by western blotting using the indicated antibodies. (<b>B</b>) siRNA mediated knock down of SPBP reduces p62 expression level. HeLa cells transfected with two various SPBP siRNAs were analysed by western blotting using antibodies as indicated (left panel). The graph (right panel) shows the fold reduction calculated and correlated to actin in three independent experiments with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>C</b>) Knock down of SPBP reduces the amount of p62 mRNA transcripts. The p62 mRNA levels were measured by quantitative RT-PCR. Hela cells were transfected with SPBP siRNAs or Control siRNA. RT-PCR reactions were run on p62, GADPH and β-actin mRNA. The average amount of p62 mRNA correlated to β-actin and GADPH mRNA based on two independent experiments are shown with standard deviations (*p<0.05, <i>n.s.</i> not significant). (<b>D</b>) p62 body formation upon sulforaphane treatment is reduced in SPBP siRNA knock down cells. HeLa cells were transfected with SPBP siRNA or Control siRNA and treated with 20 µM sulforaphane for 8 hours two days post transfection. Cells were fixed, stained with polyclonal antibodies against SPBP (green) and p62 (red) and analysed by confocal microscopy. The graph shows counting of p62 bodies in cells, each based on counting of more than 60 cells. Arrowheads indicate some of the p62 bodies in the cytoplasm.