Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 2. Ovulation rate declines with increasing body weight but is improved by treatment with ER stress inhibitors. (A) Hematoxylin and Eosin-stained ovary sections from 14-week-old gonadotropin-treated lean (+/+ or +/bbb) and obese Blobby (bbb/bbb) mice. Arrows indicate unruptured follicles, which are prevalent in ovaries from obese mice. Representative examples from n=4 mice per genotype are shown. Panels a-c are higher magnifications of the indicated follicles of the Blobby mouse. (B) The number of ovulated oocytes in 14-week-old lean and obese mice treated with saline vehicle (Veh.), salubrinal (Sal) or BGP-15 i.p. once per day for 4 days. Values are means+s.e.m. (lean mice+Veh, n=19; lean+Sal, n=5; lean+BGP-15, n=10; obese mice+Veh, n=22; obese+Sal, n=16; obese+BGP-15, n=14). Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test; P<0.0001. Right panel: the number of ovulated oocytes in oviducts is not reduced in bbb/bbb mice prior to the onset of obesity, at 6 weeks of age (+/+; +/bbb: n=11; bbb/bbb n=4; Student’s t-test).

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Staining, Saline

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 5. Treatment with salubrinal or BGP-15 induces mtDNA replication and normalizes mitochondrial membrane potential (ΔΨm) and autophagy in oocytes from obese mice. Obese mice (bbb/bbb) or lean littermates (+/+ or +/bbb) were treated with vehicle (Veh), salubrinal (Sal) or BGP-15 i.p. once daily for 4 days, and oocytes were collected from the oviducts. (A) Live oocytes stained with JC-1, where red fluorescence indicates high ΔΨm, and green indicates low ΔΨm. (B) Ratio of red to green fluorescence, an indicator of mitochondrial activity. Data are presented as means+s.e.m., n=6-30 oocytes from four mice per group. (C) Live oocytes were assessed for autophagic vacuoles, visualized as green fluorescence. Hoechst 33342 (blue). (D) Autophagy levels were quantified as the sum total of green fluorescence within each oocyte. Data are presented as means+s.e.m., n=8-10 oocytes from three mice per group. (E) mtDNA copy number in individual oocytes collected from treated mice. Data are presented as means+s.e.m.; n=19-38 oocytes from six mice per group over three independent experiments. (F) Representative examples of oocytes from three or four mice per treatment group immunostained for TFAM or DRP1 (green) and Hoechst 33342 (blue). Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test, P<0.0001.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Membrane, Staining, Fluorescence, Activity Assay

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 6. Oocytes from obese Blobby mice exhibit impaired embryo development that is restored by treatment with salubrinal or BGP-15. Obese Blobby (bbb/bbb) mice or lean littermates (+/+ or +/bbb) at 14 weeks of age (except where indicated) were treated with vehicle (Veh; saline), salubrinal (Sal) or BGP-15 daily for 4 days. Ovulated oocytes were collected from oviducts 16 h following hCG injection and fertilized in vitro. (A) At 4 h after in vitro fertilization, many degenerate oocytes (arrows) from untreated obese mice were observed. (B) The percentage of viable oocytes presented as means+s.e.m. Embryo development was assessed on day 2 and day 5 following IVF and is presented as the mean percentage of embryos exhibiting appropriate (‘on-time’) development+s.e.m. (C) Two-cell embryos developed from viable oocytes by day 2. (D) Blastocysts developed from two-cell embryos by day 5. Lean, n=24; lean+Sal, n=7; lean+BGP- 15, n=6; Blobby, n=15; Blobby+Sal, n=6; Blobby+BGP-15, n=7 pools of oocytes from animals within same group. [C,D, right-hand panels are identical assessments of oocytes from 6-week-old (non-obese) mice. +/+, n=13; Blobby, n=10.] (E) Blastocysts were collected at day 5 and the mtDNA copy number of individual blastocysts was measured by using quantitative PCR. Data are expressed as mean copy number+s.e.m.; lean, n=22 blastocysts; lean+Sal, n=10 blastocysts; lean+BGP-15, n=7 blastocysts; Blobby, n=14 blastocysts; Blobby+Sal, n=10 blastocysts; Blobby+BGP-15, n=7 blastocysts collected from three independent IVF experiments. Different letters indicate significant differences by one-way ANOVA, Tukey’s post hoc test; P<0.0001.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Saline, Injection, In Vitro, Real-time Polymerase Chain Reaction

Journal: Development (Cambridge, England)

Article Title: Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors.

doi: 10.1242/dev.114850

Figure Lengend Snippet: Fig. 7. Altered fetal outcomes of oocytes from obese Blobby mice are alleviated after treatment with salubrinal or BGP-15. Obese mice (bbb/bbb) and lean littermates (+/+) were treated with vehicle (Veh; saline), salubrinal (Sal) or BGP-15 for 4 days, followed by the isolation of oocytes from oviducts and fertilization by using IVF. Blastocysts were collected at day 5 of culture and transferred to non-obese pseudo-pregnant recipient mice with six blastocysts per uterine horn. On embryonic day 14.5, fetuses were collected and weighed (A). Liver (B), heart (C) and kidney (D) were dissected and the relative mtDNA copy number normalized to β-actin nuclear DNA was determined. Data are presented as means+s.e.m.; lean+Veh, n=18 fetuses from six surrogates; Blobby+Veh, n=20 fetuses from six surrogates; Blobby+Sal, n=22 fetuses from six surrogates; Blobby+BGP-15, n=15 fetuses from four surrogates. (E) Liver mtDNA samples (n=4 fetuses from each treatment group) were analyzed by using next generation sequencing, and rearrangements were identified. Different letters indicate significant differences calculated by one-way ANOVA, Tukey’s post hoc test; P<0.01.

Article Snippet: Salubrinal (Invitrogen) was injected i.p. at 1 mg/kg of bodyweight (Sokka et al., 2007) in approximately 0.1 ml saline daily for four consecutive days starting the day prior to treatment with eCG.

Techniques: Saline, Isolation, Next-Generation Sequencing

Journal: Scientific Reports

Article Title: Invasive lobular and ductal breast carcinoma differ in immune response, protein translation efficiency and metabolism

doi: 10.1038/s41598-018-25357-0

Figure Lengend Snippet: LumA ILC has lower protein translation efficiency than LumA IDC. ( a ) Protein/mRNA ratio in LumA ILC (n = 115) and LumA IDC (n = 246). Mann-Whitney U test, *p < 0.05. ( b ) Protein levels of translation regulators in LumA ILC vs LumA IDC. Protein expression data were from TCGA RPPA. Limma was used to compare the protein expression of LumA ILC to LumA IDC with CPE correction. Significant DE proteins (Benjamini-Hochberg method adjusted p-value < 0.05) were marked in red (up-regulated in LumA ILC) or blue (down-regulated in LumA ILC). ( c ) Regulation network of protein translation regulators in Fig. . Modified from . ( d ) Protein synthesis rate of ILC and IDC cell lines. O-propargyl-puromycin (OPP) labeled the newly synthesized proteins. Fluorescence representing the amount of OPP indicated the protein synthesis rate of cells. Cells without OPP labeling or pre-treated with cycloheximide (CHX) to inhibit protein synthesis served as negative controls. Representative data of two independent experiments were presented. Data are mean ± s.d. of 3 replicates. Two-way ANOVA, ***p < 0.001. ( e ) Dose response and IC50 of translation inhibitors in ILC and IDC cell lines. 4EGI-1 to inhibit the binding of eIF4E and eIF4G, cycloheximide to inhibit the tRNA translocation, salubrinal to inhibit eIF-2α were used. Representative data of at least two independent experiments were presented. Data in dose response curves are mean ± s.d. of 6 replicates. Data in bar graphs of IC50 are mean + upper limit of 95% confidence intervals. Two-tailed t-test was performed to compare the IC50s between ILC and IDC cell lines. The p-values for cycloheximide, salubrinal and 4EGI-1 are 0.15, 0.17 and 0.42, respectively.

Article Snippet: Cycloheximide (C4859; Sigma-Aldrich, St. Louis, MO, USA), 4EGI-1 (S7369; Selleck Chemicals, Houston, TX, USA), and Salubrinal (SC-202332A; Santa Cruz, Dallas, TX, USA) were dissolved in DMSO (4-X; ATCC).

Techniques: MANN-WHITNEY, Expressing, Modification, Labeling, Synthesized, Fluorescence, Binding Assay, Translocation Assay, Two Tailed Test

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: iScience

Article Title: Undersized telomeres in regulatory T cells link to the pathogenesis of allergic rhinitis

doi: 10.1016/j.isci.2023.108615

Figure Lengend Snippet: Assessment of the effects of an eIF2a inhibitor on mitigating experimental AR An AR mouse model was established using the DME protocol. (A‒H) The AR response, including AR clinical symptoms (nasal itch and sneezing; A and B), allergic mediators (EPX and Mcpt1; [C and D]) and Th2 cytokines (IL-4, IL-5, IL-13; [E‒G]) in NLF, and serum sIgE (H). (I and J) AMCs were isolated from the airway tissues and analyzed by FCM. Gated FCM plots show Treg counts in CD4 + T cells (the gating strategy is the same as Figure 4 ). Boxplots show Treg counts of 6 mice per group. (K‒N) Tregs were purified from AMCs. (K and L) mRNA levels of Eif2a and Tert in Tregs. (M) Telomerase activity. (N) TL in Tregs. The data of bars are presented as mean ± SD of indicated items from 12 mice per group. Each dot in bars presents one sample. Statistics: ANDME followed by the Bonferroni test. The group labels of J–N are the same as those of I. The experiments were repeated three times. Abbreviations: NC: Naive control. AR: Allergic rhinitis. Salubrinal: An inhibitor of eIF2a in nasal instillations (20 μM). FCM: Flow cytometry. AMC: Airway mononuclear cell. DME: DME. TL: Telomere length. Treg: Regulatory T cell. NLF: Nasal lavage fluid. DMSO: Dimethyl sulfoxide (the solvent of Salubrinal; 0.2 μg DMSO/mL in nasal instillations).

Article Snippet: Salubrinal , Solarbio Life Science , .

Techniques: Isolation, Purification, Activity Assay, Control, Flow Cytometry, Solvent

Journal: iScience

Article Title: Undersized telomeres in regulatory T cells link to the pathogenesis of allergic rhinitis

doi: 10.1016/j.isci.2023.108615

Figure Lengend Snippet:

Article Snippet: Salubrinal , Solarbio Life Science , .

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Software