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. Author manuscript; available in PMC: 2014 Nov 1.
Published in final edited form as: Nat Cell Biol. 2014 Apr 6;16(5):401–414. doi: 10.1038/ncb2934

Figure 4. Cx43 internalization triggers mobilization of pre-autophagosomal structures from the plasma membrane and autophagosome biogenesis.

Figure 4

(a) Immunoblots for the indicated proteins in mouse embryonic fibroblasts (MEFs) treated with tamoxifen (Tx) or lindane (Lnd) in the absence or presence of lysosomal protease inhibitors (PI). Right: Changes in LC3-II levels relative to those detected in untreated cells (n=4 independent experiments). (b) Immunofluorescence for Cx43 and LC3 in NRK cells treated as in a in the presence or absence of chloroquine (CQ) for 3h. Right: Quantification of the average number of LC3-positive puncta per cell (n= 3 wells, 3 independent experiments, >30 cells per experiment). (c) Time course of changes in the average LC3-positive puncta per cell after addition of Lnd to control NRK cells. (d) Fluorescence images of Wt or Cx43−/− MOBs transfected with mCherry-GFP-LC3 and incubated or not with Tx or Lnd. Bottom: Quantification of autophagolysosomes (n= 3 wells, 3 independent experiments, >50 cells per experiment). (e) Immunostaining for Atg16 and Cx43 in MEFs treated with the indicated compounds. Representative images as merged channels are shown. Right: quantification of the average of cytosolic Atg16-positive puncta per cell (n= 3 wells, 3 independent experiments, >30 cells per experiment). (f) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 treated with Lnd and untreated. Single channel images in inverse black and white and merged color images are shown. Boxed areas are shown at higher magnification. Arrows: double (yellow) and triple (white) colocalization. (g, h) Immunostaining for Atg16 in Cx43 knocked down (−) (g) or knocked out (−/−) (h) cells. Right: quantification of the average of cytosolic Atg16 positive puncta per cell (n= 3 wells, 3 independent experiments, >50 cells per experiment). (i) Immunoblot for Atg16 and Atg14 in immunoprecipitates (IP) of endogenous Cx43 from MEFs wild type cells maintained in the absence of serum (4h) or incubated in presence of Tx or Lind. I: input; FT: flow through. All values are mean+s.e.m and differences with untreated cells are significant for (*) p<0.01. Bars: 5 μm. Uncropped images of blots are shown in Supplementary Fig. 9.