Figure 8. Depletion of connexins induces autophagosome formation.

(a) Immunoblot for the indicated proteins in mouse embryonic fibroblasts (MEFs) control or knocked down (−) for the three different connexins. (b) Immunofluorescence of LC3 in MEFs control (Ctr) or knocked down (−) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n= 3 wells, 3 independent experiments, >50 cells per experiment). (c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (−) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as loading control. (d) Representative images of MEFs control or knocked-down (−) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL) (n= 3 wells, 3 independent experiments, >50 cells per experiment). (e) Electron micrographs of MEFs control or knock-down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n= 4 wells, 4 independent experiments, >10 micrographs per well). (f) Immunoblot for indicated proteins in immunoprecipitates (IP) for Cx26 or Cx32 (left) or Atg16 (right) from liver homogenates. Inp: input, FT: flow through. BIP is shown as negative control for IP. All values are mean+s.e.m and significant for (*) p<0.01 (ANOVA+Bonferroni test was used in b). Bars: 5 μm in fluorescence images; 1 μm and 0.1 μm in top panels and inserts of e. Uncropped images of blots are shown in Supplementary Fig. 9.