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. 2014 Sep 22;5:4998. doi: 10.1038/ncomms5998

Figure 2. CALM modulates autophagy substrate clearance in vitro.

Figure 2

(a) p62 vesicle formation in CALM knockdown HeLa cells. Confocal pictures are shown. #CALM-downregulated cells where p62 vesicles accumulate. Data are representative of three independent experiments and shown as mean ±s.e.m. (n≥500 cells; *P<0.01; two-tailed t-test). Scale bars, 5 μm. (b) Western blot analysis of p62, actin and GAPDH in HeLa cells and HEK cells (basal conditions, BC, or without serum for 1 h) where CALM was knocked down using shRNA or siRNA, as indicated. Data are mean ±s.d (n=3 experiments for HeLa cells and HEK cells; *P<0.05; two-tailed t-test). (c) Percentage of Q74-expressing cells with aggregates in CALM knockdown HeLa cells. Data depict one representative experiments performed in triplicate, out of three independent experiments and shown as mean ±s.d. (*P<0.05; two-tailed t-test). (d) Tau-positive tangle formation in CALM knockdown cells. HeLa cells transiently expressing DsRed-tau 4R were treated with Baf A1 for 4 h as indicated. Cells were fixed and analysed by confocal microscopy after immnunostaining for phosphorylated tau using PHF1 antibody (green). Data represent the number of cells with phosphorylated tau-positive tangles as mean ±s.e.m. (n=3 experiments; *P<0.01; two-tailed t-test). Scale bars, 5 μm.