Journal: Biochimica et biophysica acta. General subjects

Article Title: Inhibiting IRE-1 RNase signaling decreases HIV-1 Tat-induced inflammatory M1 state in microglial cells.

doi: 10.1016/j.bbagen.2022.130219

Figure Lengend Snippet: Fig. 1. Effect of IRE1 inhibitor 4μ8C on cell viability and apoptotic fate of Tat-induced M1-like BV-2 cells. (A) BV-2 cells were incubated with increasing con centrations of IRE1 inhibitor 4μ8C at different time-points and cell viability was assessed by MTT assay. (B) BV-2 cells were cultured with 10 μM 4μ8C in presence or absence of 400 nM recombinant Tat for 48 h and cell viability was estimated as described above. Data expressed as percentage of untreated cells ± SD. (C) Dot plot graphs showing apoptotic cell rates as percentage of BV-2 cells incubated with 10 μM 4μ8C in presence or absence of recombinant 400 nM Tat for 48 h. Data acquired by flow cytometry after 7-AAD/Anexin V staining. Experimental controls were performed with heat-inactivated Tat (TatHI, 400 nM) and untreated cells (NT). Data shown is representative of three independent experiments. Statistical significance is indicated in relation to untreated cells group (ANOVA, p < 0.05*).

Article Snippet: The IRE1 inhibitor 4μ8C was purchased from Tocris Bioscience (Minneapolis, USA, #4479/50).

Techniques: Incubation, MTT Assay, Cell Culture, Recombinant, Flow Cytometry, Staining

Journal: Biochimica et biophysica acta. General subjects

Article Title: Inhibiting IRE-1 RNase signaling decreases HIV-1 Tat-induced inflammatory M1 state in microglial cells.

doi: 10.1016/j.bbagen.2022.130219

Figure Lengend Snippet: Fig. 2. 4μ8C downregulates HIV Tat-induced ER stress in BV-2 cells by interfering with the IRE1 pathway. (A) BV-2 cells were incubated for 48 h with 400 nM recombinant Tat with or without 10 μM 4μ8C, expression profile of IRE1 pathway-related genes was assessed by RT-qPCR and displayed as whisker-box plots of normalized expression ratio. (B) Expression of XBP-1u (unspliced form) and XBP-1s (spliced form) of Tat-treated BV-2 cells with or without 10 μM 4μ8C for 48 h evaluated by Western blotting. Bar graph shows the normalized band intensity values of Western blottings as mean ± SE. The figure was prepared from different fields of the same western blot images and this editing is indicated by the dotted line. Experimental controls were performed with heat-inactivated Tat (TatHI, 400 nM). Data are representative of three independent experiments. Statistical significance is indicated in relation to Tat-treated cells (Student’s t-test, p < 0.05*).

Article Snippet: The IRE1 inhibitor 4μ8C was purchased from Tocris Bioscience (Minneapolis, USA, #4479/50).

Techniques: Incubation, Recombinant, Expressing, Quantitative RT-PCR, Whisker Assay, Western Blot

Journal: Biochimica et biophysica acta. General subjects

Article Title: Inhibiting IRE-1 RNase signaling decreases HIV-1 Tat-induced inflammatory M1 state in microglial cells.

doi: 10.1016/j.bbagen.2022.130219

Figure Lengend Snippet: Fig. 3. Inhibition of IRE1 RNase activity attenuates Tat-induced M1 phenotype in BV-2 cells. (A) Bar graphs show the levels of secreted TNF-α, IL-6 and NO of BV-2 cells incubated with 400 nM recombinant Tat with or without 10 μM 4μ8C for 48 h. Data acquired by CBA or Griess assay and expressed as mean ± SD. (B) Flow cytometry analysis of BV-2 cells incubated with 400 nM recombinant Tat with or without 10 μM 4μ8C for 48 h and labeled with CD16/32. Expression rates (dot plots) and fluorescence measurements (histograms) are displayed. Null refers to unmarked cell control for flow cytometry assessments. Experimental controls were per formed with heat-inactivated Tat (TatHI, 400 nM) and untreated cells (NT). (C) Tat-treated BV-2 cells (400 nM) were incubated with or without 10 μM 4μ8C for 48 h, iNOs and Arg-1 genes expression was assessed by RT-qPCR and displayed as normalized expression ratio. Experimental controls were performed with heat- inactivated Tat (TatHI, 400 nM). Data are representative of three independent experiments. Statistical significance is indicated in relation to Tat-treated cells (Student’s t-test, p < 0.05*).

Article Snippet: The IRE1 inhibitor 4μ8C was purchased from Tocris Bioscience (Minneapolis, USA, #4479/50).

Techniques: Inhibition, Activity Assay, Incubation, Recombinant, Griess Assay, Flow Cytometry, Labeling, Expressing, Fluorescence, Control, Quantitative RT-PCR

Journal: International journal of molecular sciences

Article Title: CDNF Interacts with ER Chaperones and Requires UPR Sensors to Promote Neuronal Survival.

doi: 10.3390/ijms23169489

Figure Lengend Snippet: Figure 3. CDNF promotes survival of ER–stressed cultured neurons and regulates UPR signaling in vitro. (A–F) CDNF treatment downregulated the expression of UPR genes sXbp1, Atf6, and Grp78 in embryonic mouse DA neurons under thapsigargin (Tg)-induced ER stress. DA neurons were cultured 5–7 days before adding CDNF (100 ng/mL) and inducing ER stress by adding 200 nM Tg. RNA was isolated from these neurons after 24 h. The expression levels of UPR marker transcripts were determined by qPCR. Each transcript’s levels have been normalized to beta-actin housekeeping gene and presented as a fold change to respective control data set from non-treated neurons. Shown are means of n = 10–18 experiments ± S.E.M. Repeated-measures ANOVA and Sidak’s multiple comparison post hoc test. (G,H) Survival-promoting activity of CDNF against ER stress is dependent on active IRE1α and PERK pathways. Mouse SCG neurons were microinjected with (G) recombinant human CDNF protein or (H) CDNF expression plasmid and treated with 2 µM tunicamycin and 2 µM PERK signaling inhibitor GSK2606414 or 25 µM IRE1α signaling inhibitor 4µ8C or 2 µM IRE1α inhibitor KIRA6. Then, 72 hours later, the number of living injected, fluorescent neurons was counted and expressed as the percentage of initially injected neurons. Shown are the means of 3–6 experiments ± S.D. Survival percentages in CDNF plasmid- or protein-injected groups were compared to the empty vector or PBS-injected controls of the same treatment group using ordinary one-way analysis of variance (ANOVA) and Sidak’s multiple comparison post hoc test. *, **, ***, **** denote p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively.

Article Snippet: IRE1 signaling inhibitors 4μ8C (4479, Tocris Bioscience, Bristol, UK) or KIRA6 (19151, Cayman Chemical, Ann Arbor, MI, USA) or PERK signaling inhibitor GSK2606414 (516535, Merck Millipore, Burlington, MA, USA) were used when indicated.

Techniques: Cell Culture, In Vitro, Expressing, Isolation, Marker, Control, Comparison, Activity Assay, Recombinant, Plasmid Preparation, Injection

Journal: The Journal of Biological Chemistry

Article Title: The cytoprotective protein MANF promotes neuronal survival independently from its role as a GRP78 cofactor

doi: 10.1016/j.jbc.2021.100295

Figure Lengend Snippet: MANF is an UPR-regulating protein in primary neuron cultures. A , a schematic depiction of MANF expression constructs used in this study with red asterisks indicating a point mutation. The domains of MANF are shown above the pCR3.1 MANF construct scheme. Shown are also ER signal peptide (SP), the Twin-StrepII-HA tag (SH) in pre-SH-MANF constructs, and secondary structure elements: α ( green bars )- and 3 10 ( blue bars ) helices. B , mouse SCG neurons maintained in the presence of NGF were treated with tunicamycin and microinjected with ( B ) MANF expression plasmid or ( C ) recombinant MANF protein. Mouse SCG neurons maintained in the presence of NGF were microinjected with ( D ) the indicated expression plasmids or ( E ) recombinant human MANF protein, and treated with 2 μM tunicamycin and 2 μM PERK signaling inhibitor GSK2606414 or 25 μM IRE1 signaling inhibitor 4μ8C. The number of living injected, fluorescent neurons was calculated 72 h after the injections and expressed as the percentage of initially injected neurons. Shown are the means of two to six experiments ± SD MANF plasmid or protein injected groups were compared with the empty vector or PBS injected controls of the same treatment group using one-way analysis of variance (ANOVA) and Sidak’s multiple comparison post hoc test. ∗∗∗ denotes p < 0.001, ∗∗∗∗ denotes p < 0.0001. The null hypothesis was rejected at p < 0.05. F , E13 midbrain floor neuron cultures were cultured with MANF (100 ng/ml), GDNF (100 ng/ml), or no neurotrophic compound (no factor) for 5 days. Dopamine (DA) neurons were identified by tyrosine hydroxylase (TH)-immunostaining and expressed as % of cell survival in each condition compared with the positive control, GDNF-treated neurons. Shown are the means ± S.D. of five independent experiments per condition. ANOVA and Tukey’s multiple comparison post hoc test. G , MANF protein protects embryonic dopamine neurons from ER stress. Dissociated cultures of E13.5 NMRI mouse midbrain floors were grown for 5 days and then treated with 100 nM thapsigargin (Tg) for 3 days. After 3 days, the cultures were immunostained for TH. TH-positive neurons were counted and expressed as a percentage of nontreated neurons. Shown are the means of eight experiments ± SD. Tg-treated group was compared with control group and Tg, MANF group using ANOVA and Dunnett’s multiple comparison post ho c test. H , E13.5 DA neurons were cultured without any trophic factors for 5 days, then treated 3 days as indicated with Tg, MANF, IRE1 (4μ8C or KIRA6), or PERK (GSK2606414) inhibitors. The results are expressed as percentage of TH-positive cell survival as compared with the non-Tg treated condition. Data of each treatment groups were compared with Tg+MANF group, n = 5, ordinary one-way ANOVA and Sidak’s multiple comparisons post hoc test. I – N , DA neurons were cultured 5 to 7 days in vitro , then ER stress was induced by adding 200 nM thapsigargin (Tg). MANF was added to the cultures at the same time as Tg. RNA was isolated after 24 h. The expression levels of ER stress marker transcripts were normalized to levels of β-actin in the same samples. Shown are means of n = 11 to 15 experiments ±SD. ANOVA and Tukey’s multiple comparison post hoc test. ∗, ∗∗, ∗∗∗, ∗∗∗∗ denote p < 0.05, p < 0.01, p < 0.001, p < 0.0001, respectively. ER, endoplasmic reticulum; GDNF, glial cell line–derived neurotrophic factor; IRE1, inositol-requiring enzyme 1; MANF, mesencephalic astrocyte-derived neurotrophic factor; NGF, nerve growth factor; PERK, protein kinase RNA-like ER kinase; SCG, superior cervical ganglion; UPR, unfolded protein response.

Article Snippet: PERK signaling inhibitor GSK2606414 (516535, Merck Millipore) or IRE1 signaling inhibitor 4μ8C (4479, Tocris Bioscience) were used when indicated.

Techniques: Expressing, Construct, Mutagenesis, Plasmid Preparation, Recombinant, Injection, Comparison, Cell Culture, Immunostaining, Positive Control, Control, In Vitro, Isolation, Marker, Derivative Assay