The presence of GJA1-20k results in the formation of actin puncta. (A) Schematic of GJA1 plasmid designs used in overexpression experiments. Internal translation of truncated isoforms does not occur in plasmids where methionines (green) are mutated to leucines (red). (B) Representative live-cell confocal images of the actin cytoskeleton, visualized by LifeAct-mCherry, in HeLa cells expressing either 0.5 µg of GJA1-20k-GFP or GST-GFP; scale bars = 5 µm. (C) Representative live-cell confocal images of the actin cytoskeleton, visualized by LifeAct-mCherry, in HeLa cells expressing either GJA1-WT (1 µg), GJA1-43k-GFP (1 µg) + GST-GFP (0.5 µg), or GJA1-43k-GFP (1 µg) + GJA1-20k-GFP (0.5 µg); scale bars = 5 µm. (D) Quantification of actin puncta density of all HeLa transfection conditions. Data are presented as mean ± SD (n = 100–200 cells per group from four independent experiments). ****P < 0.0001 by two-tailed Mann–Whitney U test (GST-GFP and GJA1-20k) or by one-way ANOVA with multiple comparisons with Bonferroni’s post hoc test (GJA1-WT, GJA1-43k, and GJA1-43k + GJA1-20k).