Methods: Immunofluorescence (IF) and biochemical fractionation were performed on transduced immortalized human esophageal epithelial cells (EPC2) stably overexpressing CAPN14 grown in submerged culture. Fractionation was also performed on untransduced EPC2 cells grown at the air-liquid interface (ALI) and on primary esophageal epithelial cells grown in submerged culture.
Results: CAPN14 was readily detectable in whole cell lysates from transduced, but not untransduced, EPC2 cells. Fractionation revealed ~77% of CAPN14 to be in the cytosolic fraction, with ~7% percentage detectable in the membrane and ~16% in the nuclear fractions in human esophageal epithelial cells grown in submerged culture. However, following differentiation into a stratified squamous epithelium, endogenous CAPN14 was mainly localized in the nucleus. IF staining of phorbol-12 myristate-13 acetate (PMA) and ionomycin treated esophageal epithelial cells in monolayer culture showed CAPN14 to undergo changes in localization with a shift from the cytoplasmic to nuclear compartments and then to the plasma membrane after 30 and 180 minutes, respectively.
Conclusions: CAPN14 is localized to the cytoplasm, membrane, and nucleus in human esophageal epithelial cells. Following cellular activation (PMA/ionomycin), CAPN14 shows a dynamic distribution, most notable by its presence in the nucleus, consistent with a key cellular function, yet to be described.