Only a limited recommendation can be made in favour of the use of statin based on current data

effectively and widely influence protein production in these cells. Several spots exhibited a 0.1- to 0.9-fold decrease in signal intensity; however, 2 major spots exhibited a 1.3-fold increase in intensity. We purified one of the spots with at least a 1.3-fold increase in signal intensity, and subsequent mass spectrometric analysis strongly suggested that this protein was HSP47. Next, we examined whether HSP47 expression fluctuated after inhibition of O-glycosylation in Colo 205 cells and NIH3T3 cells. Immunoblotting analyses using the 4 HSP47 Prevents Golgi Stress-Induced Cell Death doi: 10.1371/journal.pone.0069732.g002 anti-HSP47 antibody revealed very low levels of HSP47 in cells with or without DMSO treatment. By contrast, HSP47 protein levels increased remarkably in a dosedependent manner after GalNAc-bn treatment in both cell lines. Real-time PCR analysis confirmed the results from immunoblotting analyses. The expression of HSP47 mRNA was hardly detectable in cells with or without DMSO. However, HSP47 mRNA levels were remarkably increased after GalNAc-bn treatment. Furthermore, HSP47 mRNA and protein levels were not altered by tunicamycin or thapsigargin treatment, as Tm and Tg induce ER stress by Chebulinic acid price preventing protein N-glycosylation in the ER. We further examined whether another Golgi stressor affected the expression of HSP47. Monensin is a selective Golgi inhibitor and function by inhibiting protein transport and modification of sugar chains in the Golgi apparatus. After 12 h of monensin treatment, NIH3T3 cells exhibited a remarkable dose-dependent increase in HSP47 protein levels. These findings indicate that Golgi stress induced by not only GalNAc-bn but also monensin elicited an increase in HSP47 mRNA and protein expression in heavily Oglycosylated cells, such as Colo 205 and NIH3T3 cells. 5 HSP47 Prevents Golgi Stress-Induced Cell Death HADHA, GM130, and calnexin were used as 19219009 markers for mitochondria, Golgi apparatus, and the ER, respectively. HSP47 immunoreactivity in both untreated and GalNAc-bntreated cells strikingly overlapped with calnexin immunoreactivity, but not with HADHA or GM130 immunoreactivity. HSP47 expression maintained the normal volume of the Golgi apparatus after O-glycosylation inhibition As shown above, inhibition of O-glycosylation elevated HSP47 expression, suggesting that increased expression of HSP47 protects the Golgi apparatus during Golgi stress. Thus, to clarify the importance of HSP47 expression during the inhibition of O-glycosylation in NIH3T3 cells, we used the siRNA-based knockdown method to examine the effects of HSP47 depletion 15325591 on NIH3T3 cell viability. To this end, we established NIH3T3 cells in which HSP47 expression was suppressed by HSP47-targeted siRNA. As shown in Does HSP47 protect cells from Golgi stress We sought to determine whether the increase in the volume of the Golgi apparatus in the HSP47-knockdown cells after Golgi stress reflected hyperfunction or hypofunction. To this end, we examined the morphology of NIH3T3 cells under Golgi stress. First, we assessed the time course of the morphological changes in HSP47 siRNA-transfected cells during GalNAcbn treatment using light microscopy. No obvious alterations in morphology were observed in untransfected control cells and scrambled siRNA-transfected cells even on day 3 after GalNAc-bn treatment. However, when HSP47 siRNA-transfected cells were treated with GalNAc-bn, an apparent morphological change was identified 3 d after trea

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