Collagen dynamics were not involved in Golgi stress signaling.

<p>(A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using antibodies against type I collagen. (B) NIH3T3 cells were stained with anti-type I collagen antibodies and anti-type IV collagen antibodies at 24 h after GalNAc-bn stimulation. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells. Scale bar: 50 µm.</p

HSP47 expression protected cells from Golgi stress-induced cell death.

<p>(A) Representative phase-contrast micrographs of NIH3T3 cells. (a–c) nontransfected cells, (d–f) scrambled siRNA-transfected cells, and (g–h) HSP47 siRNA-transfected cells. GalNAc-treated cells were observed at 3 d after stimulation. Scale bar: 20 µm. (B) Viability of NIH3T3 cells 1 d after GalNAc-bn treatment (0–20 mM) as measured by a cell counting assay. Quantitative data are expressed as mean (SEM) of at least 3 independent experiments. *p <0.05 (Student’s <i>t</i> test).</p

HSP47 expression protected NIH3T3 cells from Golgi stress-induced apoptosis.

<p>(A) TUNEL-positive cells were significantly increased in HSP47 siRNA-transfected NIH3T3 cells after GalNAc-bn treatment. Results are presented as the percentage of TUNEL-positive cells over total cells, normalized to untreated controls. Data are expressed as the mean (SEM) of at least 3 independent experiments. *p < 0.05 (Student’s <i>t</i> test). (B) The expression of HSP47 or GAPDH protein in NIH3T3 cells after treatment with GalNAc-bn for the indicated periods was examined by western blot analysis using anti-HSP47 or anti-GAPDH antibodies. (C) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using antibody against caspase-2. NIH3T3 cells treated with etoposide (Eto) were used as a positive control for caspase-2 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells, NoS, no stimulated cells. (D) Western blot analysis showed a remarkable increase in Bcl-xL protein levels in the NIH3T3 cell line stably over-expressing Bcl-xL (BCL) in comparison with NIH3T3 cells expressing the pCAGGS empty vector (CAG). (E) CAG cells and BCL cells treated with GalNAc-bn for 6 h were examined by western blot analysis using an antibody against caspase-2 after HSP47 siRNA transfection.</p

The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of <i>O</i>-Glycosylation Inhibition

<div><p>The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of <i>O</i>-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of <i>O</i>-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of <i>O</i>-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.</p> </div

HSP47 expression affected mitochondria caspase-9 cleavage.

<p>(A) NIH3T3 cells treated with GalNAc-bn for the indicated periods were examined by western blot analysis using an antibody against caspase-9. NIH3T3 cells treated with staurosporine (STS) were used as a positive control for caspase-9 cleavage induction. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells; NoS, no stimulated cells. (B) CAG and BCL cells treated with STS for 12 h were examined by western blot analysis using an antibody against caspase-9. (C) Western blot analysis showed caspase-2 and GAPDH protein expression 2 d after transfection with caspase-2 siRNA. (D) CAG, BCL, and caspase-2 siRNA transfected NIH3T3 (Cas2 siRNA) cells treated with GalNAc-bn for 24 h were examined by western blot analysis using an antibody against caspase-9 after HSP47 siRNA transfection.</p

HSP47 expression affected mitochondria cytochrome <i>c</i> efflux.

<p>(A) NIH3T3 cells were fractionated into cytoplasmic (Cyto) and mitochondrial (Mito) fractions, which were analyzed by western blotting using antibodies against HADHA (mitochondrial marker) and β-tubulin (cytoplasmic marker). (B) Western blot analysis showed cytochrome <i>c</i> protein levels in each of the Cyto and Mito fractions for 2 d after transfection with scrambled or HSP47 siRNAs of NIH3T3 cells treated with GalNAc-bn for the indicated periods. Cont, nontransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells. (C) Quantification of the intensity of protein bands from GalNAc-bn-treated samples of (B). Cytochrome <i>c</i> release from the mitochondrial to the cytoplasmic fraction in HSP47 siRNA-transfected NIH3T3 cells at 24 h after GalNAc-bn stimulation. Data are presented as the relative efflux ratio (Mito/Cyto) compared to that of the corresponding untreated control group, which was set at 1.0. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells. (D) Western blot analysis showed cytochrome c protein levels of the Cyto and Mito fractions for 2 d after transfection with HSP47 siRNAs of CAG and BCL cells at 24 h after GalNAc-bn stimulation.</p

HSP47 was shown to be a regulator of Golgi volume in NIH3T3 cells.

<p>(A) Western blot analysis showed HSP47 and GAPDH protein expression 3 d after transfection with scrambled or HSP47 siRNAs. Cont, nontransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells. (B) NIH3T3 cells were stained with anti-GM130 antibodies with (d–f) or without (a–c) GalNAc stimulation. GalNAc-treated cells were observed at 24 h after stimulation. (f) High magnification images of a part of each photograph are shown in the white windows. Scale bar: 20 µm. (C) Results of the quantification of the volume of the Golgi apparatus in NIH3T3 cells. Golgi volume in NIH3T3 cells was measured using ImageJ software. The results are expressed as the mean (SEM) of at least 3 independent experiments. *p < 0.05 (Student’s <i>t</i> test). Cont, nontransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells.</p

Golgi stress-induced elevation of HSP47 protein expression.

<p>(A, B) Western blotting analysis showed a remarkable and dose-dependent increase in HSP47 protein levels after GalNAc-bn stimulation (A, Colo 205 cells; B, NIH3T3 cells). (C) Quantification of the protein bands in NIH3T3 cell samples after 1-d treatment with GalNAc-bn, Tm, or Tg. Data are expressed as mean (SEM) of at least 3 independent experiments. ***p < 0.01 (Student’s <i>t</i> test). Tm, tunicamycin; Tg, thapsigargin. (D) Western blotting analysis showed a dose-dependent increase in HSP47 protein levels after monensin stimulation for 12 h in NIH3T3 cells.</p

Golgi stress induced Golgi disassembly in HSP47 siRNA-transfected NIH3T3 cells.

<p>Representative electron micrographs of NIH3T3 cells. (A) Electron micrographs of NIH3T3 cells at 2 d after transfection with scrambled or HSP47 siRNAs and 3 d after treatment with DMSO or GalNAc-bn. GalNAc-bn treatment induced numerous vacuoles around the Golgi apparatus. Cont, untransfected cells; Scr, scrambled siRNA-transfected cells; siRNA, HSP47 siRNA-transfected cells. N, nucleus; g, Golgi apparatus; m, mitochondria; c, primary cilium. Scale bar: 4 µm. (B) Electron micrographs of NIH3T3 cells at 2 d after transfection with HSP47 siRNA and 3 d after treatment with GalNAc-bn. There are 3 nuclei in 1 cell and numerous vacuoles extending into the cytoplasm. N, nucleus. Scale bar: 20 µm. (C) Selection of high magnification images of the small rectangles in panel C. Numerous vacuoles were observed not only around the Golgi apparatus but also in the cytoplasm. g, Golgi apparatus; m, mitochondria. Scale bar: 4 µm.</p

Effects of inhibition of ERK, Akt, PKA, CREB and GPRC6A on the proliferation of l-arginine-treated fibroblasts.

<p>Fibroblast proliferation was measured by MTS assay after pretreatment with 0 or 6l-arginine and subsequent treatment with 10 μM U0126, LY294002, H-89, siRNA CREB and siRNA GPRC6A. Results are expressed as the mean ± SEM of 3 independent experiments.</p