Fig. 1.

Knockdown of Sec22B impairs autophagic flux. (A–C) HeLa cells were transfected for 72 hours with 20 nM of control siRNA or siRNA against Sec22B. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A₁ (Calbiochem) was added to the cells in the last 4 hours before harvesting. The graphs in B and C report the quantitative analysis of LC3-II and p62 levels relative to actin in three independent experiments performed at least three times in triplicate. The P values for the densitometric analyses were determined by factorial ANOVA test using STATVIEW v4.53 (Abacus Concepts), where the control condition was set to 100. The y-axis values are shown as a percentage and the error bars denote s.e.m. (n=3; ***P<0.001; **P<0.01; NS, non-significant). (D) HeLa cells seeded on glass coverslips were transfected for 96 hours with 20 nM of either anti-Sec22B or control siRNA. In the last 48 hours, cells were retransfected with the same siRNA mix plus 2 μg of the GFP–HD74 expression vector. The P values for assessing aggregation of EGFP–HDQ74 were determined using Student's t-test (n=3; ***P<0.001).