Growth-dependent localization of SAC1 in fibroblasts. (A) Confocal immunofluorescence microscopy of human fibroblasts grown in the presence of 15% FCS. The cells were fixed, permeabilized, and costained with polyclonal anti-SAC1 (green) and monoclonal anti-PDI or anti-TGN46 antibodies (red). Bar, 15 μm. (B) Human fibroblasts were cultivated for 24 h in the presence of 0.5% FCS to induce quiescence. The quiescent cells were subsequently stimulated with 15% FCS for another 24 h. Cells were processed for confocal immunofluorescence microscopy as in A. Bar, 15 μm. (C) NIH3T3 cells were grown in 10% NBS and then starved in the presence of 0.5% serum for the indicated times. SAC1 was visualized by immunofluorescence microscopy. The ratio of mean fluorescence intensity in Golgi regions (F_Golgi) to the mean fluorescence intensity in ER structures (F_ER) was calculated at different time points and used as a measure of SAC1 distribution. Values are means ± SD from individual cells (n = 7–12). (D) NIH3T3 cells were starved in 0.5% serum for 48 h and then stimulated with 10% serum. SAC1 was visualized by immunofluorescence analysis. The ratio between mean fluorescence intensity in the Golgi regions (F_Golgi) and mean fluorescence intensity in ER-structures (F_ER) was calculated at different time points and used as a measure of SAC1 distribution. Values are means ± SD from individual cells (n = 10–15).