tment, although inhibition of HSP47 expression alone failed to induce the morphological change. SB-366791 site GalNAc-bn treatment of HSP47 siRNA-transfected cells elicited cell shrinkage or a cell deathassociated morphology. We postulated that if HSP47 repression plays a key 
role in this morphological change following GalNAc-bn treatment, recovery of HSP47 expression in HSP47 knockdown NIH3T3 cells should prevent cell shrinkage. Thus, 2 d after HSP47 siRNA transfection, NIH3T3 cells were retransfected with empty vector control or a human HSP47 construct harboring neutral mutations in the siRNA oligonucleotide sequence. Cell numbers of empty vector control and hHSP47expressing NIH3T3 cells decreased in a dose-dependent manner after GalNAc-bn treatment. However, siRNAtransfected cells overexpressing hHSP47 exhibited an approximately 2-fold increase in cell number compared to NIH3T3 cells transfected with the empty vector control. Collectively, our findings indicate that HSP47 expression may play a role in protecting NIH3T3 cells from Golgi stressinduced cell death. Without GalNAc-bn treatment, the ultrastructure of the Golgi apparatus was similar in control cells with or without DMSO treatment, untransfected control cells, scrambled siRNA-transfected cells, and HSP47 siRNA-transfected cells. The Golgi apparatus in these cells consisted of curved parallel arrays of flattened cisterns expanding at their lateral ends. These arrays were rather compactly located in the perikarya, and a number of small vesicles were associated with these arrays. One day after GalNAc-bn treatment, a small number of vacuoles appeared around the Golgi apparatus in untransfected control cells, scrambled siRNA-transfected cells, and HSP47 siRNAtransfected cells. Three days after GalNAc-bn treatment, a small number of vacuoles were still observed around the Golgi apparatus in untransfected control cells and scrambled siRNA-transfected 19219009 cells. By contrast, HSP47 siRNA-transfected cells exhibited a marked increase in the number of vacuoles, many of which were located around the Golgi apparatus, at 3 d after GalNAcbn treatment. In addition, in some cells, these vacuoles dispersed into the cytoplasm, and in a few cases, most of the cytoplasm was occupied by the vacuoles. GalNAc-bn treatment of siRNA HSP47-transfected cells caused an evident change in the Golgi apparatus after 3 d. The curved parallel arrays were disassembled and dispersed more widely in the perikarya of these cells than in control cells. Many of the disassembled curved parallel arrays were associated with areas close to the inner and outer surfaces of the arrays, a part of the disassembled curved parallel arrays in the cytoplasm between different parallel arrays. Furthermore, consistent with 6 HSP47 Prevents Golgi Stress-Induced Cell Death doi: 10.1371/journal.pone.0069732.g003 7 HSP47 Prevents Golgi Stress-Induced Cell Death doi: 10.1371/journal.pone.0069732.g004 8 HSP47 Prevents Golgi Stress-Induced Cell Death light microscopic observations, a few cells exhibited split nuclei with condensed chromatin. However, formation of apoptotic bodies was not observed. Therefore, the increase in Golgi volume observed at the light microscopic level after Golgi stress may be attributed to the disassembly of the Golgi apparatus. HSP47 expression protected 15866179 cells from Golgi stressinduced apoptosis As described above, GalNAc-bn treatment of HSP47 knockdown cells induced cell shrinkage; disassembly of the Golgi apparatus; appearance of
