(A) SV-589 cells were set up on day 0 at 7.5 × 104 cells/well of six-well plates with glass coverslips in medium A containing 10% FCS. On day 1, the cells were switched to identical medium or medium A containing 10% NC-LPPS, 10 µM compactin, and 50 µM mevalonate as indicated. Following incubation for 16 hr at 37°C, the cells were treated in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol for an additional 4 hr at 37°C. The cells were subsequently fixed and subjected to immunostaining as described in the legend to Figure 5. Coverslips were mounted using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Images were obtained with a Zeiss Axio Observer Epifluorescence microscope using a 63× oil Plan-Apochromat objective and Ziess Axiocam color digital camera in black and white mode. For the purpose of presentation, brightness levels were adjusted across the entire image using ImageJ software (National Institution of Health, USA). (B) SV-589 cells were set up on day 0 at 3 × 104 cells/well of a twelve-well plate with glass coverslips. On day 1, the cells were transfected using FuGENE6 with 200 ng DsRed-Golgi (Clontech Laboratories, Inc., Mountain View, CA); the total amount of DNA/well was adjusted to 500 ng by the addition of empty pcDNA3.1 vector. 4 hr after transfection, cells received a direct addition of medium A containing 10% FCS or 10% NC-LPPS supplemented with 10 µM compactin and 50 µM mevalonate (final concentrations) as indicated. Following incubation for 16 hr at 37°C, cells were treated for 4 hr in the absence or presence of 30 µM geranylgeraniol (GGOH), 30 µM farnesol (FOH), 1 µg/ml 25-HC, or 10 µg/ml cholesterol. Cells were subsequently fixed and subjected to immunostaining and imaging as described in (A). Shown are cropped images representing a 64 × 64 micron-portion of the original images 219 × 174 microns in size.