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Figure 5 Disease Mechanisms: Role of CHGA in Catecholamine Storage Vesicle Formation

Effect of small interfering RNA (siRNA) to "silence" expression of CHGA protein in sympathoadrenal PC12 cells. PC12 cells were grown after transfection with the indicated amount of 22-bp siRNA-rat CHGA duplexes at 4 µg/well for 72 h. (A) Visualization of CHGA immunoreactivity by chemiluminescent immunoblot. Whole cell lysates were prepared, and expression of CHGA and actin (control, "housekeeping" protein) was evaluated by SDS-PAGE followed by immunoblotting using a rabbit polyclonal anticatestatin (rat CHGA _367-387) antibody or a monoclonal antiactin primary antibody. (B) Densitometry to quantify CHGA and actin protein expression, using NIH image v1.6 software. (C and D) Electron microscopy. Ultrastructural examination of the effect of siRNA CHGA "silencing" on dense-core granule biogenesis in sympathoadrenal PC12 cells. Cells were grown in the presence of 22-bp siRNA-rat CHGA duplexes (D), or mock/control (C) at 4 µg/well for 72 h. Aldehyde-fixed cells were processed for electron microscopy as described in the Methods section. Dense core granules (arrowheads) are seen either "docked" to, or in the vicinity of the plasma membrane. Note that fewer secretory granules are present in the cytoplasm of siRNA-rat CHGA-treated cells. (C) Control (mock-treated) PC12 cells. (D) CHGA siRNA-treated PC12 cells. Scale bar: 500 nm. (E) Quantification of the abundance of dense-core secretory granules reveals a decreased number of granules per cell planes, as defined by the number of granules found in an XY section of the midcell body. n = 40 for both populations of cells. ***p < 0.0001, by t test. er = endoplasmic reticulum; m = mitochondria; n = nucleus.