Journal:

Article Title: PKR Regulates B56?-mediated BCL2 Phosphatase Activity in Acute Lymphoblastic Leukemia-derived REH Cells *

doi: 10.1074/jbc.M800951200

Figure Lengend Snippet: eIF2α and AKT promote B56α protein expression. A, Western blot analysis was performed to determine the effect of salubrinal on B56α expression. HA antibody was used to detect HA-GFP protein in REH/GFP cells and exogenous HA-B56α protein in REH/WT B56α cells on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 75 μm salubrinal for 24 h. Antibodies against eIF2α, p-eIF2α, and tubulin were used as controls. B, Western blot analysis was performed to determine the effect of LY294002 on B56α expression in REH cells transfected with control shRNA and REH cells transfected with PKR shRNA. Rabbit polyclonal antibody against B56α protein was used on total lysates (1 × 106 cell equivalents) from untreated cells or cells treated with 1 μm LY294002 for 24 h. Antibodies against p-AKT, AKT, and tubulin were used as controls.

Article Snippet: Where appropriate, PKR inhibitor (Calbiochem), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}} LY294002 (Calbiochem), LiCl (Sigma), salubrinal (Tocris, Evansville, MO), and/or MG-132 (Sigma) was added to cells.

Techniques: Expressing, Western Blot, Transfection, Control, shRNA

Journal: Scientific Reports

Article Title: HIF-1α triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis

doi: 10.1038/s41598-018-36063-2

Figure Lengend Snippet: ER stress is involved in hypoxia-induced alveolar epithelial cells apoptosis. Primary rat AECs were placed in normoxia (Nx) (21% of O 2 ) or exposed to hypoxia (Hx) (1.5% of O 2 ) for increasing times (4–24 h). Protein levels of ATF4 ( A ), ATF6α/ATF6 ratio ( B ) and XBP1s/XBP1 ratio ( C ) were evaluated by western blotting. Quantification has been done on at least 5 independent experiments, representing the densitometry analysis of each proteins of interest reported to β-actin. Primary rat AECs were transfected with plasmids coding for luciferase reporter activity of ATF4 (the amino acid response element: AARE) ( D ) or ATF6α/XBP1s (the endoplasmic reticulum response element: ERSE) ( E ) and exposed to hypoxia for 6 h. Primary rat AECs were placed in normoxia or exposed to hypoxia for 24 h in the presence or absence of ER stress modulators salubrinal (SLB, 100 µM) or 4-phenylbutyrate (4-PBA, 100 mM). The activity of effector caspase 3 was evaluated by enzymatic assay ( F ), and expression of the pro apoptotic marker Bim was evaluated by RT-qPCR ( G ). n = at least 5 independent experiments. Data were submitted to a Kruskal-Wallis one-way analysis of variance followed by a Dunn’s multiple comparison tests, except for AARE and ERSE activity data submitted to a Mann-Whitney analysis. * P < 0.05, and ** P < 0.01: significantly different from control value in normoxic cells. # P < 0.05: significantly different from the value in untreated hypoxic cells. ns: non-significant difference between normoxic condition and hypoxic condition.

Article Snippet: 100 μM salubrinal (Santa Cruz) or 100 mM 4-phenylbutyrate (4-PBA) (Sigma) were used to inhibit UPR pathways.

Techniques: Western Blot, Transfection, Luciferase, Activity Assay, Enzymatic Assay, Expressing, Marker, Quantitative RT-PCR, Comparison, MANN-WHITNEY, Control

Journal: Scientific Reports

Article Title: HIF-1α triggers ER stress and CHOP-mediated apoptosis in alveolar epithelial cells, a key event in pulmonary fibrosis

doi: 10.1038/s41598-018-36063-2

Figure Lengend Snippet: CHOP is involved in hypoxia-induced alveolar epithelial cells apoptosis. Chop mRNA expression was evaluated by RT-qPCR in primary rat AECs placed in normoxia (Nx) (21% of O 2 ) or exposed to hypoxia (Hx) (1.5% of O 2 ) for increasing times (4-24 h) ( A ). Chop mRNA expression was evaluated by RT-qPCR in primary rat AECs treated with 100 μM salubrinal (SLB) or 100 mM 4-phenylbutyrate (4-PBA) and exposed 6 h to hypoxia (1.5% of O 2 ) ( B ). A549 cells were transfected with CHOP siRNA or scrambled (scr) siRNA. 24 h after transfection, A459 cells were placed for 24 h in hypoxia (0.5% of O 2 ). CHOP silencing was validated by evaluation of CHOP expression by RT-qPCR ( C ) and western blotting ( D ). Expression of the targeted CHOP pro-apoptotic marker CHAC-1 was evaluated by RT-qPCR in transfected A549 ( E ). A549 cells were transfected with an empty GFP vector (+GFP) or a plasmid coding for CHOP-GFP protein fusion (+CHOP). Transfection efficiency was evaluated by western blotting ( F ). Activity of effector caspase 3 ( G ) and BIM mRNA expression ( H ) were evaluated after 24 h exposure to hypoxia (0.5% of O 2 ). n = at least 5 independent experiments. Data were submitted to a Kruskal-Wallis one-way analysis of variance followed by a Dunn’s multiple comparison tests, except for caspase 3 activity and BIM mRNA expression data submitted to a Mann-Whitney analysis. * P < 0.05, ** P < 0.01 and *** P < 0.001: significantly different from normoxic control value ( A , B ), from value in normoxic cells transfected with scrambled si-RNA ( C – E ) or from GFP-transfected cells value ( G , H ). # P < 0.05 significantly different from value in untreated hypoxic cells (B) or from value in hypoxic cells transfected with scrambled siRNA ( C – E ).

Article Snippet: 100 μM salubrinal (Santa Cruz) or 100 mM 4-phenylbutyrate (4-PBA) (Sigma) were used to inhibit UPR pathways.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Marker, Plasmid Preparation, Activity Assay, Comparison, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

doi: 10.1101/2021.02.01.429116

Figure Lengend Snippet: A, B, WT and Atf6b −/− hippocampal neurons were infected with a control or CRT-expressing lentiviral vector, and the expression levels of CRT and calnexin were measured by western blotting (A). n=3 experiments. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01 by a two-way ANOVA followed by the Bonferroni tests. Cells were then treated with Tm (1μg/ml) for 24 h, and cell death was evaluated by immunocytochemical staining for cleaved caspase-3 (B). Scale bar: 20 μm. n=3 experiments. Data are shown as mean ± SEM. *p < 0.05, ***p < 0.001 by a two-way ANOVA followed by the Bonferroni tests. C, WT and Atf6b −/− hippocampal neurons were treated with Tm (1μg/ml) together with BAPTA-AM (5μM), 2-APB (2μM) or salubrinal (5μM). Cell death was evaluated by immunocytochemical staining for cleaved caspase-3. n=3 experiments. Typical images are shown in Figure S5 Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p< 0.001 by a two-way ANOVA followed by the Bonferroni tests.

Article Snippet: In some cases, 2-APB (12 μM, 0.5 μl in total; FUJFILM Wako Pure Chemical Co., Osaka, Osaka, Japan) or salubrinal (1mg/kg; Cayman Chemical, Ann Arbor, MI, USA) was co-injected with KA into the hippocampus, or intraperitoneally injected 30 min before KA administration, as previously described ( Sokka et al. , 2007 ; Kim et al. , 2014 ; Ikebara et al. , 2017 ).

Techniques: Infection, Expressing, Plasmid Preparation, Western Blot, Staining

Journal: bioRxiv

Article Title: The ATF6β-calreticulin axis promotes neuronal survival under endoplasmic reticulum stress and excitotoxicity

doi: 10.1101/2021.02.01.429116

Figure Lengend Snippet: Brain sections including the CA3 area of the hippocampus obtained from WT and Atf6b −/− mice at 3 days after injection with KA, KA plus 2-APB and KA plus salubrinal were subjected to Nissl staining (A) or immunohistochemical staining for cleaved caspase-3 (B). The right graphs depict the number of surviving CA3 neurons (A) and cleaved caspase-3-positive cells (B), respectively. n=6 mice. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 by a two-way ANOVA followed by the Bonferroni tests.

Article Snippet: In some cases, 2-APB (12 μM, 0.5 μl in total; FUJFILM Wako Pure Chemical Co., Osaka, Osaka, Japan) or salubrinal (1mg/kg; Cayman Chemical, Ann Arbor, MI, USA) was co-injected with KA into the hippocampus, or intraperitoneally injected 30 min before KA administration, as previously described ( Sokka et al. , 2007 ; Kim et al. , 2014 ; Ikebara et al. , 2017 ).

Techniques: Injection, Staining, Immunohistochemical staining