Several biopharmaceutical proteins fused to Zera remain encapsulated in PB-like organelles in transfected mammalian cells. (A) Western blots of Zera-Ct, Zera-hGH and Zera-EGF demonstrating accumulation inside transfected mammalian cells (lanes 1) without significant secretion into the cell culture media (lanes 2). (B-D) Immunodetection of Zera-Ct in 293T (B), Cos1 (C) and CHO (D) transfected cells using an anti-calcitonin antibody and FITC-labeled secondary antibody. Green fluorescence indicates recombinant protein accumulation in PB-like organelles in all three cases. (E, F) Electron microscopy images of a CHO cell line stably transfected with Zera-hGH; (E) CHO cell showing a PB-like organelle (PB) containing electron-dense structures surrounded by a classical rough-ER membrane (see ribosomes in inset). (F) Immunodetection of Zera-hGH recombinant protein in PBs induced in transfected CHO cells. Cryosections were incubated with an anti-hGH antibody and labeled with Protein A-gold particles (10 nm). PB, protein body-like organelle; M, mitochondria; ER, endoplasmic reticulum. (G) Viability of CHO cells stably transfected with Zera-ECFP and Zera-EGF. Relative percentages of living, necrotic and apoptotic transfected cells were compared to those of non-transfected controls (CHO WT). (H) Proliferation profiles of CHO cells expressing Zera-hGH and Zera polypeptides as compared to non-transfected CHO WT cells over 1 to 4 days of incubation.