In primary fibroblasts, endogenous procollagen colocalizes with Hsp47 in the ER. Confocal images of cells after incubation in the presence of 500 µg·ml⁻¹ ascorbate (A and B) or 50 µg·ml⁻¹ ascorbate (C) for 0 (images i and ii) and 30 min (images iii and iv), respectively. (A and B) Maximum intensity projection images of z stacks through primary adult skin fibroblasts NHDF-Ad. (C) Shows single z slice images of nontransformed foreskin fibroblasts (BJ-5ta). Cells were labeled using antibodies against COL1A1 (green) and either Hsp47 (A) or the COPII marker Sec31A (B and C) in red, as well as a cis-Golgi marker GRASP65 (blue). Panels show whole cells, as well as corresponding enlargements of the Golgi area on the right. Enlargements show the separate channels in gray scale as well as the overlay image including nuclear staining in magenta (DAPI, imaged as separate channel). Arrows highlight punctate COL1A1 structures with high signal intensity localizing in close proximity to the Golgi. Scale bars, 10 µm; in enlargements, 1 µm. The NHDF-Ad show varying expression levels of Hsp47 and COL1A1 (A, images i–iv). Localization of COL1A1 to punctate structures in the Golgi area occurs at both 0 and 30 min in the presence of ascorbate (A, images ii and iv, and B, images i–iv, arrows). The BJ-5ta show lower levels of COL1A1 expression compared with NHDF-Ad, but accumulation of COL1A1 in the Golgi area can also be observed at both time points (C, images i–iv, arrows). n ≥ 10 in each case.