p irf7  (Cell Signaling Technology Inc)

Journal: Neural Regeneration Research

Article Title: The cGAS-STING-interferon regulatory factor 7 pathway regulates neuroinflammation in Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01684

Figure Lengend Snippet: Forward and reverse sequences of each gene analyzed by real-time polymerase chain reaction

Article Snippet: They were then blocked using 5% goat serum and 0.3% Triton X-100 for 1.5 hours at room temperature (approximately 30°C), followed by incubation overnight at 4°C with the following primary antibodies: rabbit anti-IRF7 (1:200, Boster, Cat# BM4484, RRID: AB_3076601), rabbit anti-p-IRF7 (1:200, Bioss, Cat# bs-3196R, RRID: AB_10857567), and rabbit anti-CD68 (Boster, Cat# BA3638, RRID: AB_2813855).

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: The cGAS-STING-interferon regulatory factor 7 pathway regulates neuroinflammation in Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01684

Figure Lengend Snippet: Validation of the expression levels of the hub genes in an MPTP-induced mouse model of PD. (A) RT-PCR analysis of the mRNA expression levels of the five identified hub genes ( IRF7 , TAC1 , FGF13 , IFNA2 , and SOCS3 ) in striatal tissue from the MPTP-induced PD group and the saline group ( n = 6 per group). (B) Receiver operating characteristic analysis showcasing the diagnostic potential of IRF7, TAC1, and FGF13 in predicting PD. (C) Expression levels of TH, a marker gene indicative of dopaminergic neurons, in the PD and normal groups. (D) Correlation plot illustrating the negative relationship between the expression levels of the central genes IRF7 and TH ( r < –0.3 indicates a negative correlation). (E) Western blot showing the protein expression levels of TH, IRF7, and its phosphorylated form, p-IRF7, in the striatum of MPTP-treated mice ( n = 6 per group). (F) IHC staining for IRF7 in the striatum and quantitative analysis of the MOD ( n = 3 per group). IRF7 expression (brown staining) was significantly upregulated in the MPTP group. Scale bars: 100 μm (10× magnification) and 20 μm (40× magnification). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test). AUC: Area under the curve; FGF13: fibroblast growth factor 13; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IFNA2: interferon α2; IHC: immunohistochemical; IRF7: interferon regulatory factor 7; MOD: mean optical density; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin; ns: not significant; PD: Parkinson’s disease; RT-PCR: real-time polymerase chain reaction; SOCS3: suppressor of cytokine signaling 3; TAC1: tachykinin 1; TH: tyrosine hydroxylase.

Article Snippet: They were then blocked using 5% goat serum and 0.3% Triton X-100 for 1.5 hours at room temperature (approximately 30°C), followed by incubation overnight at 4°C with the following primary antibodies: rabbit anti-IRF7 (1:200, Boster, Cat# BM4484, RRID: AB_3076601), rabbit anti-p-IRF7 (1:200, Bioss, Cat# bs-3196R, RRID: AB_10857567), and rabbit anti-CD68 (Boster, Cat# BA3638, RRID: AB_2813855).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Saline, Diagnostic Assay, Marker, Western Blot, Immunohistochemistry, Staining, Immunohistochemical staining, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: The cGAS-STING-interferon regulatory factor 7 pathway regulates neuroinflammation in Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01684

Figure Lengend Snippet: Analysis of IRF7 expression, immune cell infiltration, and mitochondrial function in a mouse model of PD and in BV2 cells. (A) Box plots depicting the differential distribution of 10 immune cell types in the PD and normal groups. (B) Spearman correlation analysis between cell types with differential infiltration levels and IRF7 expression in PD. A correlation coefficient greater than 0.3 indicates a positive correlation, while a coefficient less than 0.3 suggests no significant correlation. (C) Immunofluorescence staining of striatal tissue from an MPTP-induced PD mouse model showing CD68 (red: Cy3), IRF7 (green: FITC), and DAPI (blue). Quantitative analysis of IRF7 and CD68 co-localization in 10 representative brain sections. The number of CD68 and IRF7 double-positive cells per field of vision was higher in the MPTP group than in the saline group. Scale bars: 20 μm. (D) Cellular immunofluorescence staining showing IRF7 (red: Cy3) and DAPI (blue). The relative mean fluorescence intensity of the control group (red: IRF7) was significantly lower than that of the MPP + group (red: IRF7). Co-localization of IRF7 and cell nuclei. Scale bars: 20 μm. (E) Western blot analysis of the expression levels of mitochondrial function–associated proteins in MPP + -treated BV2 cells ( n = 6 independent experiments). (F) RT-PCR analysis showing the expression of mitochondrial damage–associated genes (PGC1A and TFAM) in BV2 cells treated with MPP + . Data are presented as mean ± SEM ( n = 6 independent experiments). * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IF: immunofluorescence; IRF7: interferon regulatory factor 7; MPP + : 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin; PD: Parkinson’s disease; PGC1A: peroxisome proliferator-activated receptor-gamma coactivator-1alpha; RT-PCR: real-time polymerase chain reaction; TFAM: mitochondrial transcription factor A.

Article Snippet: They were then blocked using 5% goat serum and 0.3% Triton X-100 for 1.5 hours at room temperature (approximately 30°C), followed by incubation overnight at 4°C with the following primary antibodies: rabbit anti-IRF7 (1:200, Boster, Cat# BM4484, RRID: AB_3076601), rabbit anti-p-IRF7 (1:200, Bioss, Cat# bs-3196R, RRID: AB_10857567), and rabbit anti-CD68 (Boster, Cat# BA3638, RRID: AB_2813855).

Techniques: Expressing, Immunofluorescence, Staining, Saline, Fluorescence, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Neural Regeneration Research

Article Title: The cGAS-STING-interferon regulatory factor 7 pathway regulates neuroinflammation in Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01684

Figure Lengend Snippet: The cGAS-STING pathway plays a pivotal role in regulating IRF7 activation and nuclear translocation. (A) Western blot analysis of the expression of key molecules (cGAS, p-STING, and TBK1) in the cGAS-STING-TBK1 pathway within the striatum of MPTP-treated mice ( n = 6 per group). (B, C) BV2 cells were pre-treated with either RU.521 (2 µM) or H151 (5 µM) for 12 hours before being exposed to MPP + for an additional 24 hours, and western blotting was performed to determine the relative protein levels of p-IRF7 and IRF7 in these cells ( n = 6 independent experiments). (D) Fluorescence staining for nuclei (blue: DAPI) and p-IRF7 (red: Cy3). p-IRF7 localization to the nuclei was assessed by fluorescence microscopy. The percentage of p-IRF7 nuclear translocation in the MPP + -induced group was significantly higher than that in the control group, and pre-treatment with H151 significantly reduced the percentage of MPP + -induced p-IRF7 nuclear translocation. Arrows indicate nuclear translocation; scale bars: 20 μm. The percentage of cells with significant IRF7 nuclear translocation was calculated from multiple randomly selected fields of view (control samples: three fields of view, at least 200 cells; MPP + stimulated samples: three fields of view, at least 200 cells) ( n = 3 independent experiments). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test [A] or one-way analysis of variance followed by Tukey’s post hoc test [B–D]). cGAS: Cyclic guanosine monophosphate adenosine monophosphate synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H-151: a STING inhibitor; IRF7: interferon regulatory factor 7; MPP + : 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridin; ns: not significant; p-IRF7: phospho-IRF7; p-STING: phospho-STING; RU.521: a selective cGAS inhibitor; STING: stimulator of interferon genes; TBK1: TANK-binding kinase 1.

Article Snippet: They were then blocked using 5% goat serum and 0.3% Triton X-100 for 1.5 hours at room temperature (approximately 30°C), followed by incubation overnight at 4°C with the following primary antibodies: rabbit anti-IRF7 (1:200, Boster, Cat# BM4484, RRID: AB_3076601), rabbit anti-p-IRF7 (1:200, Bioss, Cat# bs-3196R, RRID: AB_10857567), and rabbit anti-CD68 (Boster, Cat# BA3638, RRID: AB_2813855).

Techniques: Activation Assay, Translocation Assay, Western Blot, Expressing, Fluorescence, Staining, Microscopy, Control, Binding Assay

Journal: Neural Regeneration Research

Article Title: The cGAS-STING-interferon regulatory factor 7 pathway regulates neuroinflammation in Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01684

Figure Lengend Snippet: Impact of IRF7 knockdown and H151 pre-treatment on microglial inflammatory responses and phenotypic markers in BV2 cells exposed to MPP + . (A) IRF7 mRNA expression in cells subjected to si-IRF7 knockdown was determined by RT-PCR, which showed an approximate 80% reduction ( n = 3 independent experiments). (B) BV2 cells were pre-treated with si-IRF7 for 60 hours and then exposed to MPP + for an additional 24 hours. Western blot analysis was used to detect the relative protein levels of IRF7 and IFN-β in BV2 cells stimulated with MPP + after si-IRF7 pre-treatment ( n = 5 independent experiments). (C) Western blotting was used to detect the relative protein levels of iNOS and TNF-α in BV2 cells stimulated with MPP + after si-IRF7 pre-treatment ( n = 5 independent experiments). (D) BV2 cells were pre-treated with H151 (5 µM) for 12 hours and then exposed to MPP + for another 24 hours. RT-PCR was used to detect markers of the M1 ( CD16 , CD32 , and CD86 ) and M2 ( ARG1 , YM1 ) phenotypes ( n = 6 independent experiments). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t -test [A] or one-way analysis of variance followed by Tukey’s post hoc test [B–D]). ARG1: Arginase 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; H-151: a STING inhibitor; IFN-β: interferon-beta; IRF7: interferon regulatory factor 7; iNOS: inducible nitric oxide synthase; MPP+: 1-methyl-4-phenylpyridinium; ns: not significant; RT-PCR: real-time polymerase chain reaction; si-IRF7: small interfering RNA targeting IRF7; TNF-α: tumor necrosis factor-alpha.

Article Snippet: They were then blocked using 5% goat serum and 0.3% Triton X-100 for 1.5 hours at room temperature (approximately 30°C), followed by incubation overnight at 4°C with the following primary antibodies: rabbit anti-IRF7 (1:200, Boster, Cat# BM4484, RRID: AB_3076601), rabbit anti-p-IRF7 (1:200, Bioss, Cat# bs-3196R, RRID: AB_10857567), and rabbit anti-CD68 (Boster, Cat# BA3638, RRID: AB_2813855).

Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: The Journal of Clinical Investigation

Article Title: SLAMF7 and SLAMF8 receptors shape human plasmacytoid dendritic cell responses to intracellular bacteria

doi: 10.1172/JCI182467

Figure Lengend Snippet: ( A – C ) CAL-1 and stably transduced (shCTRL, shSLAMF7, shSLAMF8) CAL-1 cells were infected with DsRed– S . Typhimurium (MOI 25) for 3 hours, and then processed for phospho-flow cytometry or confocal microscopy. ( A ) Heatmap showing MFI levels for p-IRF7, p–NF-κB p65, p–STAT-1, p-p38, p–ERK-1/2, p-AKT, and p-IRF3 of Salmonella -infected cells relative to mock condition. Multiple-comparison Kruskal-Wallis test followed by post hoc Dunn’s test. n = 4–5. ( B ) Column graphs showing relative MFI of selected phosphorylated proteins in infected cells. Mean ± SD. n = 5. ( C ) Confocal images (scale bars: 20 μm) (left) and quantification (right) of p–NF-κB p65 in Salmonella -infected CAL-1 and stably transduced CAL-1 cells. n = 3. One-way ANOVA followed by Dunnett’s multiple-comparison test. ( D ) Primary human pDCs were infected and analyzed as in B but, after 1 hour, SLAMF7 or SLAMF8 were engaged by cross-linking with a specific antibody or its isotype control. One donor per n ; n = 10. ( E ) Flow cytometry histogram of EAT-2 expression (fluorescence intensity [FI]) in primary human pDCs with positivity indicated by a vertical gray bar. One donor per n ; n = 3. ( F ) Left: Flow cytometry histogram of EAT-2 FI in CAL-1 cells at resting state with positivity bar. Right: Percentages of EAT-2 + CAL-1 and stably transduced cells, infected (green) or not (gray) as in B . n = 2. ( G ) CAL-1 and stably transduced CAL-1 cells were infected as in B ; after 1 hour, washes, and gentamicin addition, MitoTEMPO (100 μM) was added for 2 hours. Then cells were processed for phospho-flow cytometry. Column graphs showing relative MFI of selected phosphorylated proteins in infected cells. Mean ± SD. n = 3. Two-way ANOVA followed by Šidák’s multiple-comparison test. Only statistically significant differences are shown. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. No P value indicates not significant.

Article Snippet: After washing, intracellular staining with antibodies for phospho-proteins (p–NF-κB p65 [Ser536] clone 93H1, 3033; p-Stat1 [Tyr701] clone D4A7, 7649; p-IRF7 [Ser477] clone D7E1W, 42016; p-IRF3 [Ser396], 29047; p-AKT [Thr308], 9275; p–p38 MAPK [Thr180/Tyr182], 9211; p–p44/42 MAPK [Erk1/2] (Thr202/Tyr204) clone D13.14.4E, 4370; all from Cell Signaling Technology) was done in 1× PBS/2% BSA.

Techniques: Stable Transfection, Infection, Flow Cytometry, Confocal Microscopy, Comparison, Control, Expressing, Fluorescence