Figure 3. Cx43 domains responsible for the inhibitory effect over macroautophagy.

(a) Immunoblot for LC3 in MOB from wild type (Wt) or Cx43 null mice (Cx43^−/−) treated with vehicle or with 18α glycyrrhetinic acid (18α GA). Glyceraldehyde-3-dehydrogenae (GPD) is shown as loading control. Levels of LC3-II (left) and LC3-II flux (right) (n= 3 independent experiments). (b) Immunofluorescence for LC3 in MOB cells treated with vehicle or 18α GA. Where indicated, chloroquine (CQ) was added to block lysosomal degradation. Bottom: Quantification of the average number of LC3-positive puncta per cell (n=3 wells, 3 independent experiments, >30 cells per experiment). (c) Immunofluorescence for LC3 in Wt or Cx43^−/− MOBs cells treated with the indicated kinase inhibitors for 2 hours. Insets: Higher magnification images in inverted black and white. (d) Endogenous LC3 puncta in the same cells shown in c (n=3 wells, 3 independent experiments, >50 cells per experiment). *, differences with Wt and §, differences with untreated. (e) LC3 puncta in Cx43^−/− MOBs transfected with constructs coding for different forms of Cx43 (full length, CT, ΔCT₂₅₈ or ΔCT₂₄₅) tagged as indicated in the scheme. Left: Representative images. Merged fields (left) and LC3 channel in reverse black and white (right) are shown (images of GFP-Cx43 are shown in Fig. 2f). Right: Quantification of the average number of LC3 puncta per cell (n=3 wells, 3 independent experiments, >50 cells per experiment). (f, g) Immunoblot for the indicated proteins of the immunoprecipitates (IP) of Atg16 from MEFs transfected with GFP-tagged full length (FL-) Cx43 or flag tagged C-terminus (CT-) Cx43 (f) or ΔCT₂₅₈-Cx43 (g) I: input; FT: flow through. GAPDH and GFP alone were used as negative controls for IP and Atg5 as positive control. All values are mean+s.e.m. Differences with control were significant for (*, §) p<0.01. Bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.