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. 2012 Aug 15;23(16):3193–3202. doi: 10.1091/mbc.E12-01-0010

FIGURE 2:

FIGURE 2:

Localization of Atg16L1 protein in the neurites of PC12 cells is independent of autophagic activity. (A) Atg16L1 signals in the neurites of PC12 cells differed from the signals of autophagy-related organelles (isolation membranes and autophagosomes). PC12 cells stably expressing EGFP-ULK1 (an isolation membrane marker; green) or EGFP-LC3 (an autophagosome marker; green) under starved conditions were immunostained with anti-Atg16L1 antibody (red). Note that Atg16L1 signals in the neurites (top and bottom, arrows) were negative for both EGFP-ULK1 and EGFP-LC3, whereas some Atg16L1 dots in the cell body colocalized with EGFP-ULK1 dots and EGFP-LC3 dots (arrowheads in the insets). Insets, magnified views of the boxed area. Scale bars, 20 μm. (B) Atg16L1 signals in the neurites of PC12 cells were insensitive to wortmannin treatment. PC12 cells stably expressing EGFP-LC3 were starved for 30 min in HBSS in the presence of dimethyl sulfoxide or 100 nM wortmannin, fixed with 4% paraformaldehyde, and then immunostained with anti-Atg16L1 antibody. Note that, in contrast to the control cells (top left, arrowheads), the Atg16L1 signals in the neurites persisted even under autophagy-defective conditions (bottom right, arrows), where hardly any EGFP-LC3–positive dots were observed (bottom left). Scale bars, 20 μm. (C) Inhibition of canonical autophagy by wortmannin. Top, PC12 cells were treated for 30 min with HBSS containing 100 nM wortmannin. After solubilizing the cells with 1% Triton X-100, we analyzed total cell lysates by 15% SDS–PAGE followed by immunoblotting with anti-actin antibody and anti-LC3 antibody. Bottom, PC12 cells stably expressing EGFP-LC3 were incubated for 30 min with HBSS containing 100 nM wortmannin (or dimethyl sulfoxide [DMSO] alone), and then the EGFP-LC3 dots in the cells (n > 70) were counted as described previously (Itoh et al., 2008, 2011). Under these experimental conditions, there was a marked decrease in the lipidated form of LC3 (LC3-II; a marker for autophagy; lane 2, lower band in the LC3 panel) and in LC3-dot formation (lower graph). **p < 0.01 (Student's unpaired t test).