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: The membrane was then incubated with primary antibodies, including rabbit anti-TH (1:1000, MilliporeSigma, Billerica, MA, USA, Cat# AB152, RRID: AB_390204), mouse anti-GAPDH (1:8000, Proteintech, Rosemont, IL, USA, Cat# 60004-1-IgG, RRID: AB_2107436), rabbit anti-IRF7 (1:1000, Boster, Wuhan, China, Cat# BM4484, RRID: AB_3076601), rabbit anti-phospho-IRF7 (1:1000, p-IRF7; Bioss, Beijing, China, Cat# bs-3196R, RRID: AB_10857567), rabbit anti-phospho-STING (1:1000, Affinity Biosciences, Changzhou, China, Cat# AF7416, RRID: AB_2843856), rabbit anti-cGAS (1:1000, Boster, Cat# A31676-1, RRID: AB_3076604), rabbit anti-interferon-beta (IFN-β; 1:1000, Bioss, Cat# bs-0784R, RRID: AB_10856574), rabbit anti-tank-binding kinase 1 (TBK1; 1:1000, Abcam, Cambridge, UK, Cat# ab40676, RRID: AB_776632), rabbit anti-inducible nitric oxide synthase (iNOS; 1:1000, Abcam, Cat# ab15323, RRID: AB_301857), rabbit anti-B cell lymphoma 2-associated X protein (BAX; 1:1000, Abclonal, Wuhan, China, Cat# A0207, RRID: AB_2757021), and rabbit anti-tumor necrosis factor-alpha (TNF-α; 1:1000, Boster, Cat# BA0131, RRID: AB_3076605) overnight at 4°C.
Techniques: Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Small Interfering RNA
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![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: <t>interferon-beta;</t> 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_9022/pmc11759022/pmc11759022__NRR-20-2361-g006.jpg)