Journal: Cell Death Discovery

Article Title: Combination of eribulin and anlotinib exerts synergistic cytotoxicity in retroperitoneal liposarcoma by inducing endoplasmic reticulum stress

doi: 10.1038/s41420-024-02103-2

Figure Lengend Snippet: A – C Dot plots showing the top 3 KEGG pathway enrichment results of anlotinib, eribulin, and combination group. D GSEA plot featuring protein processing in endoplasmic reticulum pathway. E ERS induced by combination therapy was validated through western blot, wherein key ERS markers were upregulated and apoptosis pathway was initiated. F Rescue experiments further validated that a synergistic effect was largely induced by ERS. After applying salubrinal, synergy scores were significantly reduced.

Article Snippet: Salubrinal was purchased from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot

Journal: Frontiers in Neuroscience

Article Title: PERK Pathway Activation Promotes Intracerebral Hemorrhage Induced Secondary Brain Injury by Inducing Neuronal Apoptosis Both in Vivo and in Vitro

doi: 10.3389/fnins.2018.00111

Figure Lengend Snippet: Effect of PERK pathway inhibition and activation on SBI following ICH in vivo . (A) Brain tissue samples were collected and eIF2α phosphorylation and ATF4 expression were detected by western blotting. Tubulin served as a loading control. Protein levels were quantified with ImageJ software. Mean values for the sham group were normalized to 1.0. Data represent mean ± SEM ( n = 6). ** p < 0.01 vs. sham; # p < 0.05 vs. indicated vehicle (one-way analysis of variance followed by the Student–Newman–Keuls post-hoc test). (B) Detection of CHOP and caspase-12 expression in sham, ICH, ICH + vehicle (GSK2606414), ICH + GSK2606414, ICH + vehicle (salubrinal), and ICH + salubrinal groups 48 h after ICH by western blotting. Data represent mean ± SEM ( n = 6). ** p < 0.01 vs. sham; # p < 0.05 vs. indicated vehicle. (C) Induction of apoptosis 48 h after ICH, as detected with the TUNEL assay. Double immunofluorescence analysis was performed with TUNEL (green) and an antibody against ATF-4 (red); nuclei were labeled with DAPI (blue). Scale bar = 30 μm. Quantitative analysis of TUNEL and ATF-4 double positive neurons in each group. Data represent mean ± SEM ( n = 6). ** p < 0.01 vs. sham; # p < 0.05 vs. indicated vehicle. (D) Detection of neuronal degradation in the cerebral cortex by FJB staining (green). Scale bar = 26 μm. Arrows indicate FJB-positive cells. FJB-positive cells/mm 2 was quantified at 48 h. Data represent mean ± SEM ( n = 6). ** p < 0.01 vs. sham; # p < 0.05 vs. indicated vehicle.

Article Snippet: Salubrinal and GSK2606414 were purchased from TargetMol (Boston, MA, USA).

Techniques: Inhibition, Activation Assay, In Vivo, Expressing, Western Blot, Software, TUNEL Assay, Immunofluorescence, Labeling, Staining

Journal: Frontiers in Neuroscience

Article Title: PERK Pathway Activation Promotes Intracerebral Hemorrhage Induced Secondary Brain Injury by Inducing Neuronal Apoptosis Both in Vivo and in Vitro

doi: 10.3389/fnins.2018.00111

Figure Lengend Snippet: Effect of PERK pathway inhibition and activation on OxyHb-induced neuronal apoptosis in vitro . Neurons were cultured with or without OxyHb for 48 h. Cells were exposed to GSK2606414 or salubrinal for 1 h before OxyHb treatment. (A) eIF2α phosphorylation and ATF4 expression in the control, OxyHb, OxyHb + vehicle (GSK2606414), OxyHb + GSK2606414, OxyHb + vehicle (salubrinal), and OxyHb + salubrinal groups were detected by western blotting. Data represent mean ± SEM ( n = 3). ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. indicated vehicle. (B) CHOP and cleaved-caspase-12 expression in each group was detected by western blotting. Data represent mean ± SEM ( n = 3). * p < 0.05, ** p < 0.01 vs. control; # p < 0.05, ## p < 0.01 vs. indicated vehicle. (C) Apoptosis in OxyHb-treated neurons at 48 h was detected with the TUNEL assay. Representative images from control, OxyHb, OxyHb + vehicle (GSK2606414), OxyHb + GSK2606414, OxyHb + vehicle (salubrinal), and OxyHb + salubrinal groups are shown. Scale bar = 20 μm. The percentage of TUNEL-positive cells was determined. Data represent mean ± SEM ( n = 3). ** p < 0.01 vs. control; # p < 0.05 vs. indicated vehicle.

Article Snippet: Salubrinal and GSK2606414 were purchased from TargetMol (Boston, MA, USA).

Techniques: Inhibition, Activation Assay, In Vitro, Cell Culture, Expressing, Western Blot, TUNEL Assay

Journal: Frontiers in Neuroscience

Article Title: PERK Pathway Activation Promotes Intracerebral Hemorrhage Induced Secondary Brain Injury by Inducing Neuronal Apoptosis Both in Vivo and in Vitro

doi: 10.3389/fnins.2018.00111

Figure Lengend Snippet: Proposed role of PERK signaling pathway in SBI after ICH. The PERK signaling pathway is activated by ICH, resulting in increased p-eIF2α and ATF4 protein levels. The consequent activation of the ER stress response induces neuronal apoptosis, which is blocked by application of the PERK inhibitor GSK2606414. On the contrary, activation of PERK downstream signaling pathway by salubrinal promotes apoptosis and reduces neuronal survival by blocking eIF2α dephosphorylation.

Article Snippet: Salubrinal and GSK2606414 were purchased from TargetMol (Boston, MA, USA).

Techniques: Activation Assay, Blocking Assay, De-Phosphorylation Assay

Journal: The Journal of Experimental Medicine

Article Title: Resetting translational homeostasis restores myelination in Charcot-Marie-Tooth disease type 1B mice

doi: 10.1084/jem.20122005

Figure Lengend Snippet: Salubrinal improves myelination in S63del DRG explant cultures and reduces demyelination in S63del mice. (A) Dorsal root ganglia (DRG) were dissected from E14 WT and S63del embryos and myelination was induced with 50 µg/ml ascorbic acid. Myelin internodes were detected with antibodies against MBP (red). Nuclei were visualized with DAPI (blue). Bar, 100 µm. (B and C) Number of myelinating internodes in WT and S63del DRG treated for 3 wk with salubrinal at the indicated concentrations; n = 25–50 fields from 6–8 DRG per condition. Error bars, SEM; **, P < 0.01; ***, P < 0.001. (D) Rotarod analysis of WT and S63del mice treated for 90 d with either salubrinal (Sal) or vehicle (V). Error bars, SEM; n = 12 mice per condition. (E) Semithin sections stained with toluidine blue from P180 sciatic nerves from WT and S63del mice treated with either vehicle (V) or salubrinal (Sal) for 150 d. S63del nerves show several demyelinated fibers (arrowhead) and onion bulbs (arrow). Nerves from 10 mice per genotype were analyzed; bar, 10 µm. (F–G) Percentage of onion bulbs and demyelinated fibers in sciatic nerves from 6-mo-old S63del mice treated with Sal for 150 d. Error bars, SEM; *, P < 0.05; **, P < 0.01 by Student’s t test. 8 microscopic fields from each mouse were analyzed, n = 10 mice per condition. (H and I). Electrophysiological analysis of NCV and F-wave latency in 6-mo-old mice (150 d of treatment with Sal or vehicle). Error bars, SEM; ***, P < 0.001; n.s. = not significant by Student’s t test. n = 12–18 nerves per condition.

Article Snippet: Salubrinal (Tocris) was reconstituted at 2 mM in PBS, 0.1% BSA with 10% DMSO, and i.p injected every alternate day for 150 d (P30–P180) at 1 mg/kg of body weight.

Techniques: Staining

Journal: Mucosal immunology

Article Title: β-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis.

doi: 10.1038/mi.2014.104

Figure Lengend Snippet: Figure 3 Targeted deletion of b-arr2 attenuated DSS-induced colitis in mice. b-Arr2 KO and WT mice were treated with 5% DSS to induce experimental colitis. (a) The disease activity index was determined at the indicated time points, as described in the Methods. *Po0.01 compared with the WT mice. (b, c) Colonoscopic imaging was obtained after colitis induction on days 0, 5, and 7. Ulcers were alleviated in b-arr2 KO mice and salubrinal-treated WT mice. Quantitative analysis of the endoscopic index also diminished in these two groups. (d) Colonic ulcers were reduced in b-arr2 KO and salubrinal- treated WT mice compared with WT mice after DSS treatment for 7 days. (e) Colonic mucosal damage was observably relieved in b-arr2 KO and salubrinal-treated WT mice compared with vehicle-treated WT mice after DSS feeding for 7 days (hematoxylin and eosin (H&E) staining: original magnification, 200). (f) Histological damage after DSS feeding for 7 days was scored after H&E staining as in e. Values in a, c, d and f are the mean±s.d. (n ¼ 6 in each group). DSS, dextran sulfate sodium salt; KO, knockout; WT, wild type; b-arr2, b-arrestin 2.

Article Snippet: Next, the cells were serum starved overnight and then treated with 10mM salubrinal (Santa Cruz).

Techniques: Activity Assay, Imaging, Staining, Knock-Out

Journal: Mucosal immunology

Article Title: β-Arrestin2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis.

doi: 10.1038/mi.2014.104

Figure Lengend Snippet: Figure 8 b-Arr2-mediated apoptosis by ER stress was dependent on PUMA signaling in vitro. (a) b-Arr2 activated a PUMA luciferase reporter; however, the ER stress inhibitor salubrinal suppressed this activation. (b) The protein levels of b-arr2, GRP78, and PUMA increased in a dose-dependent manner after TNF-a treatment in HCT116 cells. (c, d) The overexpression of b-arr2 promoted GRP78 upregulation, eIF2a phosphorylation, and caspase-4 activation. (e) Double staining of PUMA and GRP78 in HCT116 cells after treatment with the ER stress inducer tunicamycin. (f) Double staining of PUMA and TUNEL in HCT116 cells after tunicamycin treatment. ER, endoplasmic reticulum; GRP78, glucose-regulated protein 78; PUMA, p53-upregulated modulator of apoptosis; TNF-a, tumor necrosis factor-a; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; b-arr2, b-arrestin 2.

Article Snippet: Next, the cells were serum starved overnight and then treated with 10mM salubrinal (Santa Cruz).

Techniques: In Vitro, Luciferase, Activation Assay, Over Expression, Phospho-proteomics, Double Staining, TUNEL Assay

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

Journal: PLoS ONE

Article Title: Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) Is a Critical Mediator of Endoplasmic Reticulum (ER) Stress Signaling

doi: 10.1371/journal.pone.0064256

Figure Lengend Snippet: ( a ) PPC1 cells were transiently transfected with 2 independent IRAK2 siRNAs (gray bars) or scrambled controls (SC) (black bars). After 2 days, cells were cultured with 5 µM Thapsigargin (TG) or 100 µM Etoposide (Eto) for 3 hrs then total RNA was extracted. Relative levels of mRNAs encoding CHOP (left), BiP/Grp78 (middle), and IRAK2 (right) were compared by qRT-PCR and displayed as ratios relative to a housekeeping gene (cyclophilin). Data are mean±SD, n = 3. ( b ) PPC1 cells were transfected with IRAK2 siRNA (+) or scrambled (Sc) siRNA (−). After 2 days, reporter gene plasmids CHOP-Luc and Renilla-Luc were transfected. Then 4–6 hrs later, cells were cultured without (C) or with 5 µM CDDO-Im (CDDO) or TG, with or without the ER stress inhibitor Salubrinal for 6 hrs. Luciferase activity was subsequently measured, normalizing firefly Luc driven from the CHOP gene promoter relative to Renilla Luc and expressing data as fold-induction relative to untreated cells transfected with SC control siRNA (mean±SD, n = 3). ( c ) Cells were transfected with either scrambled or IRAK1 siRNAs and subsequently cultured without (−) or with (+) 5 µM TG for 3 hrs. Then, RNA was extracted and CHOP and IRAK1 mRNA expression were measured by qRT-PCR (normalized to Cyclophilin). Data are expressed as fold-induction relative to untreated SC control transfected cells (mean±SD, n = 3).

Article Snippet: Salubrinal (ID-5747990, 3-phenyl-N-(2, 2, 2-trichloro-1-{[(8-quinolinylamino) carbonothioyl] amino} ethyl) acrylamide) was obtained from ChemBridge (San Diego).

Techniques: Transfection, Cell Culture, Quantitative RT-PCR, Luciferase, Activity Assay, Expressing

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Guanabenz Repurposed as an Antiparasitic with Activity against Acute and Latent Toxoplasmosis

doi: 10.1128/AAC.01683-15

Figure Lengend Snippet: Impacts of guanabenz and salubrinal on in vitro bradyzoite cysts. (A) Schematic representation of the experiment. Pru strain bradyzoite cysts were generated using alkaline medium and CO2 starvation for 6 days. On day 6, guanabenz (GA), salubrinal (SAL), or vehicle (DMSO, the solvent for salubrinal) was added to the cultures, and tissue cysts were evaluated 3 days later. (B) Representative images of in vitro-generated cysts. The LDH2-GFP reporter (green) was used to visualize bradyzoites, and Dolichos biflorus lectin (DB; red) stain was used to observe cyst walls. Tz, tachyzoite; Bz, bradyzoite.

Article Snippet: Salubrinal was synthesized in collaboration with the Department of Chemistry and Chemical Biology, Indiana University–Purdue University at Indianapolis, with the assigned standard nomenclature IUSC-12447-000-A, and was stored at −20°C.

Techniques: In Vitro, Generated, Solvent, Staining