Figure 4.

Down-regulation of NHE8 results in an accumulation of enlarged electron-dense MVBs. (A) HeLa M-cells were treated with NHE8 siRNA using a double-knockdown protocol and processed for transmission EM. The resultant phenotype was characterized by an accumulation of enlarged electron-dense MVBs compared with mock-treated cells (B). C) Quantitation of the proportion of cytosol occupied by electron-lucent or electron-dense MVBs in mock or NHE8 siRNA-treated cells. Electron-dense MVBs in NHE8 siRNA-treated cells were immunolabeled with antibodies to LAMP1 (D) or LBPA (E) followed by protein A–15 nm gold on ultrathin cryosections. HeLa M-cells expressing NHE8-HA possess MVBs with morphology indistinguishable from mock-transfected cells (F), and expression of NHE8-HA rescues the dense MVB phenotype in cells depleted of NHE8 by treatment with siRNA (G). Scale bars, (A and B, F and G) 500 nm; (D and E) 200 nm.