nitd008  (MedChemExpress)

Journal: PLOS Pathogens

Article Title: A zebrafish-based in vivo model of Zika virus infection unveils alterations of the glutamatergic neuronal development and NS4A as a key viral determinant of neuropathogenesis

doi: 10.1371/journal.ppat.1012756

Figure Lengend Snippet: (A) Schematic experimental design. Cell medium (mock), DENV viral particles (serotype 2, strain 16681s) or ZIKV viral particles (strain H/PF/2013) were microinjected in the zebrafish yolk at 2 hours post-fertilization (hpf). This schematic was created with BioRender.com . (B) Survival curve over 3 days post-fertilization (dpf) of mock-infected ( n = 84), ZIKV-infected ( n = 104), ZIKV-infected and treated with 100 μM NITD008 for the whole period ( n = 94), and DENV-infected ( n = 89) larvae. ( N = 3) **** P ≤ 0.0001. Log-rank test. (C) Representative pictures of microinjected larvae at 3 dpf. ZIKV infection induced both mild and severe developmental phenotypes. (D) Quantification of the proportion of larvae with the different morphological phenotypes illustrated in (C). Visual assessment of zebrafish morphology was done based on the criteria listed in . (No injection, n = 64; Mock, n = 54; ZIKV, n = 67; ZIKV+NITD008 (100 μM), n = 62; DENV, n = 55. N = 3). Data are shown as means ± SEM. *** P ≤ 0.001; ** P ≤ 0.01; 2-way ANOVA. (E) Head size at 3 dpf of the larvae from (D) (No injection, n = 24; Mock, n = 20; ZIKV, n = 38; ZIKV+NITD008 (100 μM), n = 25; DENV, n = 26. N = 3). Data are shown as means ± SEM. **** P ≤ 0.0001; ns = not significant; one-way ANOVA. (F-G) Total ZIKV RNA (F) and ZIKV negative strand (-) RNA (G) levels in whole larvae pools (6–15 larvae) at 1, 2 and 3 dpf were determined using ddPCR. Absolute RNA copies per fish per day post-fertilization are shown. N = 3. Data are means ± SEM. *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05; Student’s t-test for each day. (H-I) Treatment of ZIKV-infected fish with 100 μM NITD008 decreased the viral load. Viral RNA copies (normalized to the number of larvae) are shown for each independent experiment in (H). Dash lines indicate results from the same independent experiment. In (I), data were normalized to the corresponding ZIKV value for each experiment. Mock, n = 40; ZIKV, n = 40; ZIKV+NITD (100 μM), n = 40. N = 4. Data are shown as means ± SEM. *** P ≤ 0.001; ** P ≤ 0.01; one-way ANOVA. n represents the number of fish; N represents the number of independent experimental repeats.

Article Snippet: Four hours post-injection, fish were either treated with 100 μM NITD008 (Tocris Small Molecules) or 0.5% DMSO as control.

Techniques: Infection, Injection

Journal: PLOS Pathogens

Article Title: A zebrafish-based in vivo model of Zika virus infection unveils alterations of the glutamatergic neuronal development and NS4A as a key viral determinant of neuropathogenesis

doi: 10.1371/journal.ppat.1012756

Figure Lengend Snippet: (A) Schematic experimental setup and behavioral analysis (created with BioRender.com ). (B) Representative swimming tracks of control (mock), ZIKV-infected (ZIKV), and ZIKV-infected and NITD008-treated (ZIKV+NITD008 (100 μM)) larvae at 4 days post-fertilization. (C) The distance moved by the larvae was assessed using the DanioVision device (Mock, n = 40; ZIKV, n = 45; ZIKV+NITD008 (100 μM), n = 30. N = 3). Data are shown as median ± 95 CI. **** P ≤ 0.0001; Kruskal-Wallis test. (D) TUNEL staining at 2 days post-fertilization showing cell death in the developing brain following injection in zebrafish embryos. A = anterior; P = posterior. Scale bars = 50 μm. (E) The number of TUNEL+ cells was quantified (Mock, n = 15, N = 3; ZIKV normal, n = 20, N = 3; ZIKV mild/severe, n = 26, N = 3; ZIKV+NITD008 (100 μM), n = 14, N = 2). Data are shown as median ± 95 CI. **** P ≤ 0.0001; Kruskal-Wallis test. n indicates the number of fish; N represents the number of independent experimental repeats.

Article Snippet: Four hours post-injection, fish were either treated with 100 μM NITD008 (Tocris Small Molecules) or 0.5% DMSO as control.

Techniques: Control, Infection, TUNEL Assay, Staining, Injection

Journal: iScience

Article Title: Dengue virus and Zika virus alter endoplasmic reticulum-mitochondria contact sites to regulate respiration and apoptosis

doi: 10.1016/j.isci.2024.111599

Figure Lengend Snippet: ERMC proteins negatively regulate mitochondrial respiration and viral replication (A–C) Huh7.5 cells were transduced with lentiviruses expressing shRNAs which target the indicated proteins (MOI = 4). Four days later, cells were trypsinized, counted, and processed for measurements of various parameters of the oxygen consumption rate using the Seahorse technology as described in the section. Data were normalized to the mean basal OCR of the non-target shRNA (shNT) control conditions as in B–4E. ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 4–5. (D) Cells were transduced as in (a-c). Two days post-transduction, cells were infected with DENV-R2A reporter viruses which express Renilla reniformis luciferase (Rluc) at an MOI of 0.01. Two days post-infection, the luciferase activity was measured as a readout of viral replication and normalized to the shNT control condition. ∗∗∗: p ≤ 0.001; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 6–10. (E–F) Two days post-transduction, cells were infected with DENV 16681s (E) or ZIKV H/PF/2013 (F) at a MOI of 0.1). 48 h later, the infectious titers of extracellular viral particles were determined by plaque assays. All values were normalized to the shNT condition. ∗∗: p ≤ 0.01; ∗: p ≤ 0.05; Kruskal-Wallis test; N = 3–6. (G and H) Primary human monocytes were transduced with lentiviruses encoding shNT, shSYNJ2BP or shRRBP1. 2 days post-transduction cells were infected with either DENV 16681s or ZIKV H/PF/2013 at an MOI of 1 in the presence of the panflaviviral anti-E antibody to enhance the infection. One day post-infection cells were collected and analyzed for their content in (G) RRBP1 and SYNJ2BP mRNAs and (H) viral RNAs using RT-qPCR. As control conditions, monocytes were treated with 10 μM of the NS5 RNA polymerase inhibitor NITD008 to demonstrate a productive replication in these cells. Mean values with standard deviations are shown.

Article Snippet: NITD008 , Tocris Small Molecules , 6045/1.

Techniques: Transduction, Expressing, shRNA, Control, Infection, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: iScience

Article Title: Dengue virus and Zika virus alter endoplasmic reticulum-mitochondria contact sites to regulate respiration and apoptosis

doi: 10.1016/j.isci.2024.111599

Figure Lengend Snippet:

Article Snippet: NITD008 , Tocris Small Molecules , 6045/1.

Techniques: Produced, Virus, Recombinant, Software

Journal: iScience

Article Title: Dengue virus and Zika virus alter endoplasmic reticulum-mitochondria contact sites to regulate respiration and apoptosis

doi: 10.1016/j.isci.2024.111599

Figure Lengend Snippet: ERMC proteins negatively regulate mitochondrial respiration and viral replication (A–C) Huh7.5 cells were transduced with lentiviruses expressing shRNAs which target the indicated proteins (MOI = 4). Four days later, cells were trypsinized, counted, and processed for measurements of various parameters of the oxygen consumption rate using the Seahorse technology as described in the section. Data were normalized to the mean basal OCR of the non-target shRNA (shNT) control conditions as in B–4E. ∗: p ≤ 0.05; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 4–5. (D) Cells were transduced as in (a-c). Two days post-transduction, cells were infected with DENV-R2A reporter viruses which express Renilla reniformis luciferase (Rluc) at an MOI of 0.01. Two days post-infection, the luciferase activity was measured as a readout of viral replication and normalized to the shNT control condition. ∗∗∗: p ≤ 0.001; ∗∗: p ≤ 0.01; Kruskal-Wallis test; N = 6–10. (E–F) Two days post-transduction, cells were infected with DENV 16681s (E) or ZIKV H/PF/2013 (F) at a MOI of 0.1). 48 h later, the infectious titers of extracellular viral particles were determined by plaque assays. All values were normalized to the shNT condition. ∗∗: p ≤ 0.01; ∗: p ≤ 0.05; Kruskal-Wallis test; N = 3–6. (G and H) Primary human monocytes were transduced with lentiviruses encoding shNT, shSYNJ2BP or shRRBP1. 2 days post-transduction cells were infected with either DENV 16681s or ZIKV H/PF/2013 at an MOI of 1 in the presence of the panflaviviral anti-E antibody to enhance the infection. One day post-infection cells were collected and analyzed for their content in (G) RRBP1 and SYNJ2BP mRNAs and (H) viral RNAs using RT-qPCR. As control conditions, monocytes were treated with 10 μM of the NS5 RNA polymerase inhibitor NITD008 to demonstrate a productive replication in these cells. Mean values with standard deviations are shown.

Article Snippet: As replication control, NS5 inhibitor NITD008 (Tocris Small Molecules) was added at a final concentration of 10μM.

Techniques: Transduction, Expressing, shRNA, Control, Infection, Luciferase, Activity Assay, Quantitative RT-PCR