Journal: Diabetologia

Article Title: Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells.

doi: 10.1007/s00125-011-2139-z

Figure Lengend Snippet: Fig. 2 Relationship between nitric oxide production and abundance of ER stress markers in response to different combinations of cytokines in MIN6 beta cells. MIN6 cells were incubated for 24 h in the absence or presence of different combinations of IL-1β (100 U/ml), IFN-γ (250 U/ml) and TNF-α (100 U/ml) as indicated. a Levels of nitric oxide in medium were determined by Griess reaction. b Western blot was performed on protein extracts for PERK, JNK and EIF2α phosphorylation (p) and CHOP. Total (t) JNK, EIF2α and β-actin served as loading controls. Representative images are shown. ***p<0.001 for cytokine effect

Article Snippet: The following antibodies were used (1:1,000 dilution unless otherwise indicated): (1) CHOP (sc-575) and total eukaryotic translation initiation factor 2 α subunit (EIF2α) (sc-11386) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); (2) phospho-PERK (Thr980, 16 F8, 3179), phospho-EIF2α (Ser51, 9721), phospho-JNK (Thr183/ Tyr185, 9251), phosphor-c-JUN (Ser73, 9164S) and total JNK (9252) (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Incubation, Western Blot, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: A functional role for Smad7 in sustaining colon cancer cell growth and survival

doi: 10.1038/cddis.2014.49

Figure Lengend Snippet: Smad7 knockdown in CRC cells enhances eIF2 α phosphorylation and downregulates CDC25A expression. ( a ) Transfection of HCT-116 cells with Smad7 antisense oligonucleotide (AS) reduces CDC25A protein expression. HCT-116 cells were either left untreated (Untr) or transfected with Smad7 AS or Smad7 sense oligonucleotide (S). After 24 h cells were washed with PBS and cultured for further 24 h. CDC25A, CDC25B and CDC25C expression was assessed by Western blotting. One of three representative experiments in which similar results were obtained is shown. ( b ) Smad7 AS treatment does not reduce CDC25A RNA expression. HCT-116 cells were either left untreated (Untr) or transfected with Smad7 AS or S. CDC25A transcripts were evaluated by real-time polymerase chain reaction. Levels are normalized to β -actin. Values are mean±S.D. of three experiments. Smad7 AS-transfected cells versus Smad7 S-transfected cells, * P <0.001. ( c ) CDC25A protein downregulation in cells treated with Smad7 AS is not reverted by proteasome inhibitors (MG115 and MG132). Representative Western blots for CDC25A and β -actin in extracts of HCT-116 cells transfected with either Smad7 S or AS and then cultured in the presence or absence of MG115 and MG132 for 24 h. One of three representative experiments in which similar results were obtained is shown. ( d ) Treatment of CRC cells with Smad7 AS enhances eIF2 α phosphorylation and this phenomenon precedes the downregulation of CDC25A expression. Representative Western blots for p-eIF2 α Ser51, eIF2 α , CDC25A and β -actin in extracts of HCT-116 cells transfected with either S or AS and then cultured for the indicated time points. One of 3 three representative experiments in which similar results were obtained is shown. ( e ) Treatment of CRC cells with Smad7 AS reduces de novo protein synthesis. Representative western blots for puromycin and β -actin in extracts of HCT-116 cells transfected with either Smad7 S or AS and then cultured for 24 h. Where indicated, cells were pulsed with puromycin. One of three representative experiments in which similar results were obtained is shown

Article Snippet: Colonic cryosections of Apc min/+ mice were stained either with hematoxylin and eosin (H&E) or with Smad7 (Biorbyt), CDC25A (Biorbyt) and p-eIF2 α Ser51 (Cell Signaling Technology).

Techniques: Expressing, Transfection, Cell Culture, Western Blot, RNA Expression, Real-time Polymerase Chain Reaction

Journal: Cell Death & Disease

Article Title: A functional role for Smad7 in sustaining colon cancer cell growth and survival

doi: 10.1038/cddis.2014.49

Figure Lengend Snippet: Inhibition of Smad7 with the specific Smad7 antisense oligonucleotide (AS) reduces the proliferation of neoplastic cells in human CRC explants. Representative pictures of SMAD7-, PCNA-, p-eIF2 α Ser51- and CDC25A-stained sections of freshly obtained CRC explants treated with either Smad7 sense oligonucleotide (S) or AS for 24 h. Isotype control stainings are also indicated. The scale bars are 40 μ m. The scale bars in the insets are 10 μ m. One of five representative experiments in which similar results were obtained is shown

Article Snippet: Colonic cryosections of Apc min/+ mice were stained either with hematoxylin and eosin (H&E) or with Smad7 (Biorbyt), CDC25A (Biorbyt) and p-eIF2 α Ser51 (Cell Signaling Technology).

Techniques: Inhibition, Staining

Journal: Cell Death & Disease

Article Title: A functional role for Smad7 in sustaining colon cancer cell growth and survival

doi: 10.1038/cddis.2014.49

Figure Lengend Snippet: Smad7 downregulation significantly reduces colonic tumorigenesis in Apc min/+ mice. ( a ) Orally administered fluorescent-labeled Smad7 antisense oligonucleotide (AS) in Apc min/+ mice is taken up by both epithelial and lamina propria mononuclear cells in the colon after 8 h of administration. No relevant staining is seen in mice treated with PBS (CTR). The scale bars are 40 μ m. The scale bar in the inset is 10 μ m. One of three separate experiments is shown. ( b ) Left panels show representative endoscopic pictures and H&E-stained sections of colon tumors developed in mice treated with either Smad7 sense oligonucleotide (S) or AS. The scale bars are 100 μ m. Graphs show the number of lesions and the endoscopic scoring of tumors developed in mice treated with either Smad7 S or AS. Data indicate mean±S.E.M. of three independent experiments in which five mice per group were considered ( n =15). ( c – e ) Representative images showing Smad7- ( c ), CDC25A- ( d ) and p-eIF2 α Ser51-positive cells ( e ) in colonic sections taken from Apc min/+ mice treated with either Smad7 S or AS. The scale bars are 20 μ m. One of five representative experiments in which similar results were obtained is shown. NT, nontumoral area; T, tumoral area

Article Snippet: Colonic cryosections of Apc min/+ mice were stained either with hematoxylin and eosin (H&E) or with Smad7 (Biorbyt), CDC25A (Biorbyt) and p-eIF2 α Ser51 (Cell Signaling Technology).

Techniques: Labeling, Staining

Journal: Pediatric research

Article Title: Enteral Leucine Supplementation Increases Protein Synthesis in Skeletal and Cardiac Muscles and Visceral Tissues of Neonatal Pigs through mTORC1-dependent Pathways

doi: 10.1038/pr.2011.79

Figure Lengend Snippet: A bundance of eIF4E·4EBP1 (A) and eIF4E·eIF4G (B), and phosphorylation of eIF2α (C) and eEF2 (D) in longissimus dorsi muscle of piglets fed LP (□), LP+L (■), or HP ( ) diets. Values are means + SEM, n = 7–10 per treatment. Means without a common symbol differed, p < 0.05.

Article Snippet: Primary antibodies were PKB (total and Ser 473 , Cell Signaling Technology Inc., Danvers, MA), mTOR (total and Ser 2448 , Cell Signaling), PRAS40 (Total and Thr 246 , Cell Signaling), 4EBP1 (total, Bethyl Laboratories Inc., Montgomery, TX and Thr 70 , Cell Signaling), eIF4G (total and Ser 1180 , Cell Signaling), S6K1 (total and Thr 398 , Cell Signaling), eIF2α (Total and Ser 51 , Cell Signaling), and eEF2 (Total and Thr 56 , Cell Signaling).

Techniques: Phospho-proteomics

Journal: Pediatric research

Article Title: Enteral Leucine Supplementation Increases Protein Synthesis in Skeletal and Cardiac Muscles and Visceral Tissues of Neonatal Pigs through mTORC1-dependent Pathways

doi: 10.1038/pr.2011.79

Figure Lengend Snippet: Phosphorylation of eEF2 and eIF2α in other muscles and visceral tissues of piglets fed a LP, LP+P, or HP diet for 24 h.

Article Snippet: Primary antibodies were PKB (total and Ser 473 , Cell Signaling Technology Inc., Danvers, MA), mTOR (total and Ser 2448 , Cell Signaling), PRAS40 (Total and Thr 246 , Cell Signaling), 4EBP1 (total, Bethyl Laboratories Inc., Montgomery, TX and Thr 70 , Cell Signaling), eIF4G (total and Ser 1180 , Cell Signaling), S6K1 (total and Thr 398 , Cell Signaling), eIF2α (Total and Ser 51 , Cell Signaling), and eEF2 (Total and Thr 56 , Cell Signaling).

Techniques: Phospho-proteomics, Muscles

Journal: Molecular medicine reports

Article Title: Atorvastatin attenuates atherosclerotic plaque destabilization by inhibiting endoplasmic reticulum stress in hyperhomocysteinemic mice.

doi: 10.3892/mmr.2016.4975

Figure Lengend Snippet: Figure 4. Effects of atorvastatin on endoplasmic reticulum stress activation in aortic lesions of hyperhomocysteinemic apolipoprotein E‑deficient mice. Immunohistochemical analysis detected a significant decrease in phosphorylated (p)‑protein kinase RNA‑like endoplasmic reticulum kinase (PERK), p‑eukaryotic initiation factor 2α (eIF2α) and glucose‑regulated protein 78 (GRP78) immunostaining in the atorvastatin group, as compared with the positive staining of these markers in the methionine group. Magnification, x200. Data are presented as the mean ± standard deviation. *P<0.05 vs. the methionine group.

Article Snippet: Rabbit polyclonal phosphorylated (p)-protein kinase RNA-like endoplasmic reticulum kinase (PERK) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA; cat. no. sc-32577); rabbit monoclonal antibodies against glucose-regulated protein 78 (GRP78; cat. no. 3177) and p-eukaryotic initiation factor 2α (eIF2α; cat. no. 3398) were obtained from Cell Signaling Technology, Inc.

Techniques: Activation Assay, Immunohistochemical staining, Immunostaining, Staining, Standard Deviation

Journal: Molecular medicine reports

Article Title: Atorvastatin attenuates atherosclerotic plaque destabilization by inhibiting endoplasmic reticulum stress in hyperhomocysteinemic mice.

doi: 10.3892/mmr.2016.4975

Figure Lengend Snippet: Figure 5. Effects of atorvastatin on homocysteine (Hcy)‑induced endoplasmic reticulum stress in macrophages. Data are presented as the mean ± standard deviation. *P<0.05 and **P<0.01 vs. the control group; #P<0.05 and ##P<0.01 vs. the Hcy group. p‑, phosphorylated; PERK, protein kinase RNA‑like endo plasmic reticulum kinase; eIF2α, eukaryotic initiation factor 2α; GRP78, glucose‑regulated protein 78.

Article Snippet: Rabbit polyclonal phosphorylated (p)-protein kinase RNA-like endoplasmic reticulum kinase (PERK) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA; cat. no. sc-32577); rabbit monoclonal antibodies against glucose-regulated protein 78 (GRP78; cat. no. 3177) and p-eukaryotic initiation factor 2α (eIF2α; cat. no. 3398) were obtained from Cell Signaling Technology, Inc.

Techniques: Standard Deviation, Control