Journal: bioRxiv

Article Title: Maladaptive oxidative stress cascade drives type I interferon hyperactivity in TNF activated macrophages promoting necrosis in murine tuberculosis granulomas

doi: 10.1101/2020.12.14.422743

Figure Lengend Snippet: A. Sst1S and WT BMDMs were stimulated with 10 ng/mL TNF for 24h, after which media was changed and cells were either re-stimulated with TNF or left unstimulated. Samples were harvested and qRT-PCR was performed after the first 24 h of stimulation, or 48 h after the original stimulation. One representative experiment shown out of four performed, error bars represent SD of technical replicates. Significance values in supplemental table 1 (two-way ANOVA). B. Sst1S BMDMs were treated as in (A), except upon media change at 24 h, were also treated with anti-IFNAR antibody or isotype control. Fold induction between antibody and control were compared to determine percent inhibition. Significance determined by comparison to isotype control (one-way ANOVA). C. Sst1S BMDMs were stimulated with 10 ng/mL TNF for 24 or 36 h or left unstimulated, and were treated with a TNF-blocking antibody 4, 12, or 24 h before harvest, as indicated, or left untreated as a control. qRT-PCR was performed on sample RNA to determine IFNβ induction, compared to untreated samples (dotted line). Significance determined by RM-ANOVA with multiple comparisons. D. Sst1S BMDMs were treated with TNF and analyzed by qRT-PCR as in (C), but inhibitors were added 6 h before harvest to determine the dependence of IFN on pathways downstream of TNF stimulation. Inhibitors include anti-IFNAR antibody, JNK inhibitor sp600125, Integrated Stress Response inhibitor (ISRIB), and the PKR inhibitor 2-aminopurine. E. Dependence of IFN on PKR was confirmed by use of C16, a more specific PKR inhibitor, in conjunction with its structural control. Significance determined by ratio-paired t-tests. F. Representative time-course of C16’s effect on IFN induction from 12 to 30 h of TNF treatment in Sst1S. C16 added 6 h before each harvest point. Fold induction of TNF + c16 compared to TNF only and untreated to determine % inhibition by c16. Error bars represent SD of technical replicates. G. Left: Example Western blot of Thr 446 Phospho- and total PKR with actin loading control in Sst1S. PKR is both induced and activated in Sst1S BMDMs by TNF alone at 24 h. Right: Quantification of three western blots confirming PKR activation. Densitometry by ImageJ software. Significance by RM-ANOVA with multiple comparisons. Statistical analysis: *p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001

Article Snippet: PKR inhibitor structural control (CAS # 852547-30-9) purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Control, Inhibition, Comparison, Blocking Assay, Western Blot, Activation Assay, Software

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Effect of PKR inhibitors (PRI and C16) on tunicamycin (Tm) induced activation of PKR and GSK‐3β in SH‐SY5Y cells. A. Untreated neuroblastoma cell lines showing slight staining of pPKRThr446 (green) and pGSK‐3βTyr216 (red) in the cytoplasm, without apoptotic nuclei. After 8 h of Tm (5 µg/mL) treatment, the labeling of pPKRThr451 and pGSK‐3βTyr216 and their co‐localization were increased in the cytoplasm and nuclei. Adding PRI (50 µM) or C16 (1 µM) after 8 h of Tm exposure lead to an attenuation of the activation of PKR and GSK‐3β in the cytoplasm and nucleus associated with a strong reduction of co‐localization. Horizontal bar 10 µM. B. The cell counting confirmed that PKR (50%) and GSK‐3β (20%) activated after 8 h of Tm treatment and it increases, respectively, to 75% and 80% after 16 h. C16 attenuates both nuclear activation of PKR and GSK‐3β.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Activation Assay, Staining, Labeling, Cell Counting

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Immunoblot analysis of Tm treatment in SH‐SY5Y cells with or without PRI peptide. A. A progressive activation of PKR which peaks after 4 h and GSK‐3β, with highest levels after 8 h. Both are reduced at the different time of treatment by the PRI inhibitor. PARP cleavage progressively increased over time after Tm exposure and PRI peptide exposure reduced PARP cleavage at 2, 4 and 8 h. Results were obtained from 5 independent experiments. *P < 0.05, **P < 0.01. B. Tm has opposite effect on GSK‐3β phosphorylation on tyrosine 216 and serine 9, with a reduction of the level of pGSK3βser9, partially reverses by PRI addition. Results were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. C. Analysis of prepared cytoplasmic and nuclear fractions from SH‐SY5Y cells revealed that GSK‐3β begins to be translocated into the nucleus from 4 h of Tm treatment. This translocation is maximum after 8 h and with consequent increase in the phosphorylation at 4 and 8 h. Nuclear activation is attenuated by the PRI at 4 and 8 h. The evaluation of cell fractionation was assessed for cytosolic fraction with anti‐KDEL and, for nuclear fraction with anti‐histone H3.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Western Blot, Activation Assay, Phospho-proteomics, Translocation Assay, Cell Fractionation

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Tm induces phosphorylation of tau in SH‐SY5Y cells, attenuated by PRI addition. A. Immunoblot analysis with anti‐Tau, anti‐pTau (AT8) antibodies showed that Tm induces the phosphorylation of Tau at AT8 epitope with highest levels after 4 h of treatment and this phosphorylation is reduced by the PRI inhibitor. B. Immunoblot analysis of Tau phosphorylation on three other epitopes shows that Tm leads to phosphorylation at 2 h and 4 h of AT100 and AT270 but not AT180 tau. Results were obtained from 5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Phospho-proteomics, Western Blot

Journal: Brain Pathology

Article Title: Modulation of Tau Phosphorylation by the Kinase PKR: Implications in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2010.00437.x

Figure Lengend Snippet: Immunoblot analysis of Aβ1‐42 effect on GSK‐3β, PKR and Tau phosphorylation. SH‐SY5Y cells were pretreated with or without 50 µM PRI peptide, and then exposed to Aβ for 4 h or 8 h. The blots showing that Aβ induces the phosphorylation of PKR, GSK‐3β and Tau which gradually increases after 4 and 8 h of treatment and this activation is significantly reduced (35 to 40%) by the PRI inhibitor. Results were obtained from 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: The PRI (peptide PKR inhibitor) (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA‐binding site of PKR and prevent its activation (25) .

Techniques: Western Blot, Phospho-proteomics, Activation Assay

Journal: eLife

Article Title: The switch-like expression of heme-regulated kinase 1 mediates neuronal proteostasis following proteasome inhibition

doi: 10.7554/eLife.52714

Figure Lengend Snippet:

Article Snippet: The PKR inhibitor ( ; ) (Cayman) was dissolved in DMSO and used at a final concentration of 1 μM and was added 1 hr before proteasome inhibitor treatment.

Techniques: Transfection, Construct, Ubiquitin Proteomics, Recombinant, Software, Generated, Sequencing, TaqMan Assay

Journal: Brain Pathology

Article Title: The PKR Activator PACT Is Induced by Aβ: Involvement in Alzheimer's Disease

doi: 10.1111/j.1750-3639.2011.00520.x

Figure Lengend Snippet: A, B and C immunoblot analysis of Aβ exposure and PRI peptide in SH‐SY5Y cells. PACT and pPKRthr446 progressively increased over time after Aβ1‐42 treatment with peaks after 8 h. PKR activation is decreased with PRI treatment, but not PACT expression, while full PKR and tubulin are stable. Results were obtained from five independent experiments (*P < 0.05; **P < 0.001; ***P < 0.0001). D, E and F PACT and pPKRthr446 levels are stable after nontoxic Aβ42‐1 treatment that supports specific role of Aβ1‐42 in enhanced PACT/PKR interaction.

Article Snippet: Reagents Inhibitor of PKR: The PRI (NeoMPS Polypeptide Laboratories, Strasbourg, France) can bind to the double‐stranded RNA binding site of PKR and prevent its activation (20) .

Techniques: Western Blot, Activation Assay, Expressing

Journal: Frontiers in Microbiology

Article Title: Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2

doi: 10.3389/fmicb.2021.624540

Figure Lengend Snippet: PERK and GCN2 are involved in eIF2α phosphorylation during DHAV-1 infection. (A) Screen the kinases that affect eIF2α phosphorylation. DEFs were infected with DHAV-1 at MOI of 1. After 22 h of infection, different concentrations of kinase inhibitors were added to DEFs for 2 h. Then, DEFs were harvested for immunoblot analysis with the indicated antibodies. (B) PERK inhibitor GSK2606414 inhibits eIF2α phosphorylation induced by DHAV-1. (C) GCN2 inhibitor GCN2-IN-1 inhibits eIF2α phosphorylation induced by DHAV-1. (D) PKR inhibitor C16 cannot inhibit eIF2α phosphorylation induced by DHAV-1. (E) Transfection of poly(I:C) activate PKR kinase. (F) DHAV-1 and poly(I:C) stimulate PKR transcription. Differences between two groups were analyzed using Student’s t -test and considered as significant at * p < 0.05, ** p < 0.01, and *** p < 0.001. The bands marked by asterisk (*) are non-specific proteins.

Article Snippet: C16 (PKR Inhibitor) and GSK2606414 (PERK inhibitor) were purchased from APExBIO, and GCN2-IN-1 (GCN2 inhibitor) was purchased from MCE.

Techniques: Phospho-proteomics, Infection, Western Blot, Transfection

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Effect of daily s.c. administration of a PKR inhibitor at 1 (▪) and 5 (▴) mg kg −1 in comparison with solvent control (DMSO : PBS 1 : 20) on body weight change ( A ) and tumour growth rate ( B ) in mice bearing the MAC16 tumour. A time course for the inhibition of body weight loss (▴) and tumour growth (▪) is shown in ( C ). The average weight of the soleus muscles after 5 days treatment is shown in ( D ), and the body composition is shown in ( E ). The conditions for tumour transplantation and conductance of the experiment are given in Materials and Methods. The number of mice in each group n =6. Differences from control are shown as a: P <0.05; b: P <0.01; or c: P <0.001, whereas differences from percentage inhibition of tumour volume are shown as f: P <0.001.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Comparison, Solvent, Control, Inhibition, Muscles, Transplantation Assay

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of phospho-PKR ( A ) and -eIF2 α ( B ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to . The blots for total PKR and eIF2 α were used as loading controls. The first lane (CON) used gastrocnemius muscle from an NTB control. Representative blots are shown and the densitometric analysis gives the ratio of the phospho to total forms as an average of three separate blots ( n =9). Differences from NTB control are shown as a: P <0.05 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.01.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Western Blot, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Western blots of 20S proteasome α -subunits ( A ), p42 ( B ) and myosin ( C ) in gastrocnemius muscle of mice bearing the MAC16 tumour after 5 days treatment with a PKR inhibitor (1 and 5 mg kg −1 ), as described in the legend to in comparison with values from non-tumour-bearing animal (NTB). Actin was used as a loading control. The first lane (CON) used gastrocnemius muscle from a non-tumour-bearing control. Representative blots are shown and the densitometric analysis is the average of at least three separate blots. Differences from NTB control are shown as b: P <0.01 or c: P <0.001, whereas differences from the solvent control are indicated as f: P <0.001 ( n =9).

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Western Blot, Comparison, Control, Solvent

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: ( A ) EMSA of nuclear binding of NF- κ B in gastrocnemius muscle of non-tumour-bearing mice (lane 1) or mice bearing the MAC16 tumour and treated with solvent alone (lanes 2–4) or the PKR inhibitor at 1 (lanes 5–7) or 5 mg kg −1 (lanes 8–10). Lane 11 is a positive control for NF- κ B (supplied by the manufacturer of the kit). ( B ) Densitometric analysis of the EMSA shown in ( A ), n =3. Differences from non-tumour-bearing controls are shown as c: P <0.001, whereas differences from solvent control are shown as e: P <0.01 or f: P <0.001.

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Binding Assay, Solvent, Positive Control, Control

Journal: British Journal of Cancer

Article Title: Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

doi: 10.1038/sj.bjc.6603704

Figure Lengend Snippet: Flow diagram showing how activation of PKR leads to inhibition of protein synthesis through phosphorylation of eIF2 α ; and increased protein degradation through activation of NF- κ B, which would be attenuated by a PKR inhibitor (PKRI).

Article Snippet: The PKR inhibitor and PhosphoSafeTM Extraction Reagent were from Merck Eurolab Ltd (Leicestershire, UK) and electrophoretic mobility shift assay (EMSA) gel shift assay kits were from Panomics (CA, USA).

Techniques: Activation Assay, Inhibition, Phospho-proteomics

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: In vivo, kinase PKR regulates the pro‐inflammatory and Aif1 gene expression response to ZIKV infection. CC071 mice (5–6 weeks‐old) were treated with DMSO or the inhibitor of PKR (IPKR) 1 h before and 3 days after IC inoculation of either NaCl (NI) or 10 5 FFU of ZIKV. Mice were necropsied 6 days after infection with one hemisphere used for immunofluorescence and the other one used for RNA extraction and gene expression analysis. (a–d) Levels of RNAs purified from whole brain extracts were determined by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO) and n = 11 (ZIKV + iPKR). Dot plots show means with one dot for each brain and significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p ‐values near significance are indicated. (e) Correlation analysis of the expression levels of the Il1b , C4 , and Aif1 genes (as determined in NI + iPKR and ZIKV + DMSO conditions) with either Eif2ak2 expression or ZIKV RNA expression. Symbols represent individual mice. (f) Brain sections of ZIKV‐infected mice treated either with DMSO (ZIKV + DMSO) or PKR inhibitor C16 (ZIKV + iPKR) were labeled with anti‐NeuN (specific of neurons), anti‐IBA1 (specific of microglia) and anti‐NS2B (specific of ZIKV) antibodies and with DNA labeled with DAPI. Merge images of maximum intensity projection of confocal sections (z projection) are shown. Scale bars = 10 μm.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: In Vivo, Expressing, Infection, Immunofluorescence, RNA Extraction, Purification, Quantitative RT-PCR, Comparison, RNA Expression, Labeling

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: PKR regulates the morphological transformation of microglia induced by ZIKV infection. Immunohistochemical analysis of 3 NI_iPKR, 4 ZIKV_DMSO and 7 ZIKV_iPKR mice among those analyzed in Figure . (a) Representative images of brain sections stained with an anti‐IBA1 immunoperoxidase antibody. Images obtained with a slide scanner microscope were analyzed using the QuPath software in order to assess the cell body area and the number of microglial cells in the region of the whole hippocampus (yellow selection) and the CA (green selection). (b) Enlarged representative images of microglial cells with the cell body region selected by QuPath indicated in yellow. (c) Cell body areas of independent microglial cells as determined using QuPath. Symbols represent individual microglial cells. In the case of the whole hippocampus, n = 735 (NI_IPKR), n = 1763 (ZIKV_DMSO) and n = 1988 (ZIKV_iPKR) cells. In the case of the CA region, n = 188 (NI_iPKR), n = 477 (ZIKV_DMSO) and n = 437 (ZIKV_iPKR) cells. The values were converted to log 10 . Dot plots show medians with significance assessed by the non‐parametric Mann–Whitney test with p ‐value <0.05 (*) and ns = not significant; p ‐values for ZIKV‐DMSO versus ZIKV_iPKR conditions are indicated. (d) Correlation analysis of the average cell body area of microglial cells present in each CA region with either Eif2ak2 mRNA or ZIKV RNA expression measured in the corresponding brains. Symbols represent average values per brain. (e) Symbols represent individual mice with n = 3 (NI_iPKR), n = 4 (ZIKV‐DMSO), and n = 7 (ZIKV_iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test with ns = not significant.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Transformation Assay, Infection, Immunohistochemical staining, Staining, Microscopy, Software, Selection, MANN-WHITNEY, RNA Expression, Comparison

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a main regulator of the phosphorylation of STAT1 and of IRF1 expression in microglial cells. (a–h, j) Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being (a, e) either non‐ (NI) or NDV‐infected and (c, g, j) either non‐treated (NT) or treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons. Total protein extracts were collected 5 h after NDV infection and 6 h after treatment with CMs and submitted to a Western blot analysis. (b, d, f, h) Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****), <0.05 (*), and ns = not significant. (i–k) Primary cultured neurons (PCNs) and microglial cells (PCMCs) were (i, j) either non‐ (NI) or NDV‐infected for 5 h or (k) treated with recombinant IFNB for 6 h. (i, j) Protein extracts or (k) RNAs were collected and submitted to Western blot or qPCR analysis respectively. (l) The level of expression of the Irf1 and Irf7 genes in total RNAs purified from whole brain extracts of mice described in Figure was analyzed by RT‐qPCR with respect to Hrpt1 used as reference gene. Symbols represent individual mice with n = 10 (NI + iPKR), n = 12 (ZIKV + DMSO), and n = 11 (ZIKV + iPKR). Dot plots show means with significance assessed by one‐way ANOVA Tukey's multiple comparison test. p ‐value <0.0001 (****) and ns = not significant.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Expressing, Cell Culture, Incubation, Infection, Western Blot, Comparison, Recombinant, Purification, Quantitative RT-PCR

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Kinase PKR is a major regulator of non‐infected microglia's inflammatory and phagocytic response to ZIKV‐infected neurons. Primary cultured microglial cells (PCMCs) were incubated with DMSO or the inhibitor of PKR (C16) for 1 h before being either non‐treated (NT), treated with conditioned media from non‐ (CM_NI) or ZIKV‐infected (CM_ZIKV) primary cultured neurons or ZIKV alone (NT_ZIKV) for 6 h. In Figure are shown the qPCR results obtained from three independent primary cultures of microglia treated with conditioned media collected from three independent primary cultures of neurons. RNAs were collected and gene expression analysis was carried out by RT‐qPCR with respect to Rplp0 used as reference gene for genes associated with (a) the pro‐inflammatory response, (b) IFN‐I response with the level of Ifnb1 expression present in the three ZIKV‐infected PCNs from which the CMs were collected (PCN_ZIKV 64 h) also shown, (c) DAM response. Dot plots show means with one dot for each independent experiment represented in different colors. Significance was assessed by ratio‐paired t ‐test. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Infection, Cell Culture, Incubation, Expressing, Quantitative RT-PCR, Comparison

Journal: Glia

Article Title: Protein kinase R induced by type I interferons is a main regulator of reactive microglia in Zika virus infection

doi: 10.1002/glia.24619

Figure Lengend Snippet: Endogenous IFNs‐I increase the pro‐inflammatory and phagocytic capacity of newcastle disease virus (NDV)‐infected microglia. (a–d) Primary cultures of microglia (PCMCs) were either non‐infected (NI) or infected with NDV in the presence or absence of anti‐IFNAR antibody. (a–c) Gene expression analysis using RNAs collected 5 h post‐infection were carried out by RT‐qPCR using Rplp0 as reference gene. Dot plots show means with one dot for each independent culture represented in different colors. Significance was assessed by ratio paired t test. p ‐value <0.0001 (****), <0.001 (***), <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated. (d) After 15 min incubation with C3_SRBCs, phagocytosis index was determined as in Figure . Dot plots show means with one dot for each independent experiment. Significance was assessed by two‐way ANOVA Tukey's multiple comparison test. p ‐value <0.01 (**), <0.05 (*), and ns = not significant; p ‐values near significance are indicated. (e–g) PCMCs (e, f) or PCNs (g) were incubated with DMSO or the inhibitor of PKR (IPKR) 1 h before NDV infection. Gene expression analysis using RNAs from PCMCs and PCNs, collected 5 and 8 h post‐infection respectively, were carried out by RT‐qPCR using Rplp0 as reference gene. Dot plots show means with one dot for each independent culture represented in different colors. Significance was assessed by ratio paired t test. p ‐value <0.001 (***), <0.01 (**), <0.05 (*) and ns = not significant; p‐values near significance are indicated.

Article Snippet: The inhibitor of PKR (iPKR) C16 (Merck, 527450) at a 50 μM final concentration (unless indicated otherwise) or the corresponding volume of DMSO was added directly in the culture medium for 1 h before treatment or infection as described by Pham et al. ( ).

Techniques: Virus, Infection, Expressing, Quantitative RT-PCR, Incubation, Comparison