Fig. 10.

Knockdown of syntaxin-18 does not affect autophagy. (A–C) HeLa cells were transfected for 72 hours with 20 nM of siRNA against syntaxin-18 or Sly1, or control siRNA. For the assessment of autophagy by LC3-II levels, a saturating concentration (400 nM) of Bafilomycin A₁ was added to the cells in the last 4 hours before harvesting. The graphs in B and C show the quantitative analysis of LC3-II and p62 levels relative to actin in 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 in 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 anti-syntaxin-18, anti-Sly1 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 EGFP–HDQ74 aggregation were determined using Student's t-test (n=3; **P<0.01; NS, non-significant). (E,F) HeLa cells were transfected for 72 hours with either anti-syntaxin-18, anti-Sly1 or control siRNA. The effect exerted by knockdown of syntaxin-18, Sly1 protein levels (E) and processing of cathepsin B, D and L (F), was assessed by western blot analysis. The blots reported are representative of three independent experiments. p, pro form of cathepsin; m, mature form of cathepsin; i, intermediate form (for cathepsin L).