OSER membrane biogenesis is sustained by monomeric fluorescent protein fusion expression and does not involve Climp63 and SUN proteins. A. HEK293 cells grown on coverslips were transfected with calnexin-mCherry (CNX-mCherry) construct and observed by confocal microscopy 24 hours later, as described under "Materials and methods". OSER membrane expansion was observed in almost every expressing cell (n = 14). B. HEK293 cells were co-transfected with calnexin-CFP (CNX-CFP) and Climp63-GFP constructs and processed as in A. OSER structures positive for calnexin and negative for Climp63-GFP are indicated by an arrow (number of cells showing Climp63-GFP excluded from the OSER membranes, n = 23). C-E. HEK293 cells grown on glass coverslips transfected with calnexin-CFP were cultured for 24 h and labelled with an antibody for endogenous SUN1 (C), SUN2 (D) or Nesprin-1 (E), stained with a Texas Red-conjugated secondary antibody and mounted on glass slides for confocal microscopy. All but one cell stained for SUN1 (n = 56) or SUN2 (n = 35), showed exclusion of the co-stained SUN proteins from the OSER; the calnexin/SUN colocalization observed in a single cell of each set was presumably due to fortuitous targeting of SUN proteins into the OSER structures (some of which may originate from the NE ). All Nesprin-1-stained cells cells showed its exclusion from the OSER (n = 34). Multilamellar OSER structures positive for calnexin-CFP but negative for the co-stained proteins are indicated by open arrows; solid arrows indicate SUN1-, SUN2- or Nesprin-1-stained NE (SUN1 and Nesprin-1 labelling produced intra-nuclear staining, in addition to the NE staining, under various experimental conditions). Scale bar in each image is 10 μm.