Immunofluorescent staining of TMEM43 and IC disc proteins in HL-1 cells stably expressing wild-type and mutant TMEM43.

A. Immunostaining of Plakoglobin (JUP) was predominantly at the cell-cell border in control and TMEM43-overexpressing (TMEM43-WT) cells, but this significantly decreased at cell border in mutant TMEM43-transfected cells (TMEM43-S358L), with a diffuse expression throughout the cytoplasm instead. TMEM43 (green) was seen in the cytoplasm, as small spots in the TMEM43-WT cells. Greater cytoplasmic distribution was seen in control cells and near the cell edge in the TMEM43-S358L cells. B. α-catenin labeling clearly localized to the border of the cells in control and TMEM43-WT cells but in the TMEM43-S358L cells, α-catenin was clearly located diffusely throughout the cytoplasm, with very little staining specifically at the cell border. C. Immunostaining of ZO-1 labeling showed ZO-1 localization predominantly at the cell-cell junction in control and TMEM43-WT cells, while reduced amounts of ZO-1 were observed in TMEM43-S358L cells. D. Cx43 staining in TMEM43-WT cells demonstrates strong immunoreactive signals at the periphery of the cells and in the cytoplasm which is greater than those levels seen in control cells. In TMEM43-S358L cells, there is diffuse Cx43 staining of the cells throughout the cytoplasm. Images combining the TMEM43 staining/GFP fluorescence with these 4 different proteins (Merge) are shown in the right column including nuclear DAPI staining (blue). All results are representative of three independent cell culture experiments.