Figure 1: Annexin A2 regulates autophagy.

(a) Western blot analysis of tubulin, Annexin A2 and LC3-II in HeLa where Annexin A2 was knocked down using siRNA and/or transiently expressed, as indicated. The cells were starved in HBSS and treated with bafilomycin A1 (BafA1) as indicated. (SE, short exposure; LE, longer exposure). Quantification of LC3-II/tubulin ratio is shown as mean ± s.e.m. (*P<0.05; two tail one-sample t-test). (b) Number of GFP-LC3 dots per cell in Annexin A2 transiently expressing cells. HeLa cells where Annexin A2 was transiently expressed for 24 h were fixed and subjected to microscopy. The data represent the number of GFP-LC3 dots per cell shown as mean ± s.d. (*P<0.05; two-tailed t-test; n≥50 cells per condition). Representative confocal pictures are shown. Scale bars, 5 μm. (c) Quantification the proportion of mutant huntingtin (Q74)-expressing cells with aggregates (HA-HDQ74) in Annexin A2 knockdown cells. HeLa cells transiently expressing HA-HDQ74 were fixed and subjected to microscopy after HA-HDQ74 immunostaining using an anti-HA specific antibody. Representative pictures are shown. Data are mean ± s.d. of the percentage of cells with HA-HDQ74 aggregates (n=3 experiments; *P<0.05; two tail one-sample t-test). Typically, about 25% of the control cells have aggregates. We have normalised controls to 100% to enable statistics from multiple experiments, as the control numbers vary in independent experiments. (d) Colocalization between ATG9A and LC3 in Annexin A2 knockdown HeLa cells upon starvation. Confocal pictures are presented with magnified areas showing the colocalization between ATG9A and LC3. Quantification of ATG9A and LC3 colocalization is shown on the right as Pearson’s coefficient. Data are mean±s.e.m. (n≥20 cells; *P<0.05; two tail one-sample t-test). Scale bars, 5 μm.