Endogenous GJA1-20k expression results in the formation of actin puncta. (A) Western blot comparison of exogenous (ex.) GJA1-20k-GFP in HeLa cells and endogenous (end.) Cx43 and GJA1-20k in non-transfected (NT) C33A cells. HeLa cells were either NT or transfected with 0.5, 1, or 2 µg of GJA1-20k-GFP (predicted band size for GJA1-20k-GFP = 47 kDa). (B) Representative live-cell confocal imaging of actin, visualized by LifeAct-mCherry, in a wild-type C33A cell; scale bars = 10 µm. (C) Representative live-cell confocal imaging of actin, visualized by LifeAct-mCherry, in C33A cells after treatment with either control siRNA or siRNA-targeting GJA1 alone or with post siRNA knockdown expression of 0.5 µg of either GST-GFP, GJA1-43k-GFP, or GJA1-20k-GFP; scale bars = 10 µm. (D) Quantification of actin puncta density for all C33A transfection cell conditions. Data are presented as mean ± SD (n = 50–65 cells per group from three independent experiments). ns = P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by one-way ANOVA with multiple comparisons with Bonferroni’s post hoc test. Source data are available for this figure: SourceData F2.