(a) Co-localization between ATG12 and VAMP2-HA in control and CALM knockdown HeLa cells stably
expressing VAMP2-HA.
Confocal pictures are shown with magnified areas on the right showing
co-localization between ATG12 and VAMP2 in control cells and no co-localization in
CALM knockdown cells.
Quantification of ATG12-VAMP2 co-localization is shown on the graph as the
Pearson’s coefficient (data are mean
±s.d.;*P<0.05; two-tailed t-test). Scale bars,
5 μm. (b) Western blot analysis of VAMP2, actin and LC3-II in HeLa
cells where VAMP2 was
knocked down, as indicated. The cells were starved in Hanks balanced salt
solution (HBSS) and treated with Baf
A1 as indicated. (SE, short exposure; LE, longer
exposure.) Quantification of LC3-II/actin ratio is shown. Data are
representative of three independent experiments and shown as mean
±s.d. (*P<0.05; NS, not significant, two-tailed
t-test). (c) LC3 dot counting in CALM and VAMP2 knockdown cells.
CALM or VAMP2 were knocked down in cells
expressing GFP-LC3. The cells were kept in full medium or starved in HBSS
for 4 h, fixed and subjected to automated fluorescence microscopy
to score the number of LC3 dots per cell. The number of LC3 dots per cell
(shown as mean ±s.d.) is shown on the graph for each condition
(n≥300 cells per condition; BC, basal conditions).
(*P<0.01; NS, not significant, two-tailed t-test).
(d) Number of p62 dots per cell in VAMP2 knockdown. HeLa cells where VAMP2 was knocked down were fixed
and subjected to microscopy after labelling endogenous p62 using specific antibody. The
data represent the number of p62 dots per cell shown as mean ±s.d.
(*P<0.05; two-tailed t-test;
n≥300 cells per condition). (e) Percentage of
Q74-expressing cells with aggregates in VAMP2 knockdown HeLa cells. Data are from one
representative experiments performed in triplicate, out of three independent
experiments and shown as mean ±s.d. (*P<0.05;
two-tailed t-test). (f) Western blot analysis of VAMP2, actin and LC3-II in HeLa
cells stably expressing VAMP2-HA wild type or VAMP-2-HA mutated in the
CALM-binding site at
different levels (wild-type clone 6, wt Cl6: low level; wild-type clone 11,
wt Cl11: high level; mutant clone 11, AA Cl11: low level; mutant clone 12,
AA Cl12: high level). The cells were treated with Baf A1 as indicated. (SE, short
exposure; LE, longer exposure). Quantification of LC3-II/actin ratio is
shown. Data are representative of three independent experiments and shown as
mean ±s.d. (*P<0.05; two-tailed t-test).