Fig. 1: Sustained upregulation of interferon-stimulated genes Irf9 and Ddx58 in Rnaset2−/− mouse brains. | Cell Death & Disease

Fig. 1: Sustained upregulation of interferon-stimulated genes Irf9 and Ddx58 in Rnaset2−/− mouse brains.

From: Unlocking microglia pyroptosis in a model of type I interferon-driven neuroinflammation: lessons from Rnaset2−/− mice

Fig. 1

A mRNA expression levels of Irf9 were quantified by qPCR in brain samples from Rnaset2−/− and Rnaset2+/+ mice at 3, 6, 17, and 28 weeks of age. Values remain persistently increased, with mean fold changes between five and ten, showing no temporal peaks. Mean ± SEM with ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, One-way ANOVA followed by Tukey’s multiple comparison test; n = 4 for 3 and 6 weeks, n = 5 for 17 and 28 weeks. Irf9: interferon regulatory factor 9. B mRNA expression levels of Ddx58 were quantified by qPCR in brain samples from Rnaset2−/− and Rnaset2+/+ mice at 3, 6, 17, and 28 weeks of age. Values consistently show elevated levels, with mean fold changes ranging from five to ten, demonstrating no temporal peaks. Mean ± SEM with * = p < 0.05, ** = p < 0.01, ns not significant, One-way ANOVA followed by Tukey’s multiple comparison test; n = 4 for 3 and 6 weeks, n = 5 for 17 and 28 weeks. Ddx58: DEAD-Box Helicase 58. C Western blot analysis of IRF9 in Rnaset2−/− brains compared to Rnaset2+/+ at 3 weeks of age. Blot intensity differences clearly depict increased IRF9 protein amounts. Likewise, densitometry quantification shows a substantial grey value difference of over 15,000 grey values between Rnaset2−/− and Rnaset2+/+ samples. Loading control: β-ACTIN. Mean ± SEM; n = 2 per group and time point. WT1-2: Rnaset2+/+, KO1-2: Rnaset2−/−. IRF9: interferon regulatory factor 9. D Western blot analysis of RIG-I in Rnaset2−/− brains compared to Rnaset2−/− at 3 weeks of age. Differences in RIG-I grey value number are clearly visible between Rnaset2−/− and Rnaset2+/+ protein samples. Likewise, densitometry quantification demonstrates a 15,000-fold difference in grey values between Rnaset2−/− and Rnaset2+/+ protein samples. Loading control: β-ACTIN. Mean ± SEM; n = 2 per group and time point. WT1-2: Rnaset2+/+, KO1-2: Rnaset2−/−. RIG-I: Retinoic acid-inducible gene I. E Western blot analysis of IRF9 protein amount at 6 weeks of age. Densitometry analysis of IRF9 signal intensities reveals a significant increase in IRF9 protein amounts in Rnaset2−/− brain protein samples compared to samples from Rnaset2+/+ (10-fold increase in Rnaset2−/−, p < 0.001). Loading control: β-ACTIN. Mean ± SEM with *** = p < 0.001, Student’s t test; n = 4 per group and time point. WT1-4: Rnaset2+/+, KO1-4: Rnaset2−/−. IRF9: interferon regulatory factor 9. F Western blot analysis of RIG-I at 6 weeks of age. Densitometry analysis of RIG-I signal intensities depicts a significant upregulation in RIG-I protein amounts in Rnaset2−/− brains compared to Rnaset2+/+ (nine-fold increase in Rnaset2−/−, p < 0.001). Loading control: β-ACTIN. Mean ± SEM with *** = p < 0.001, Student’s t-test; n = 4 per group and time point. WT1-4: Rnaset2+/+, KO1-4: Rnaset2−/−. RIG-I: Retinoic acid-inducible gene I. G Western blot analysis of IRF9 at 17 weeks of age. Densitometry quantification of IRF9 signal intensities reveals a significant increase in IRF9 in Rnaset2−/− brains compared to Rnaset2+/+ (ten-fold increase in Rnaset2−/−, p < 0.001). Loading control: β-ACTIN. Mean ± SEM with *** = p < 0.001, Student’s t test; n = 4 per group and time point. WT1-4: Rnaset2+/+, KO1-4: Rnaset2−/−. IRF9: interferon regulatory factor 9. H Western blot analysis of RIG-I at 17 weeks of age. In comparison to Rnaset2+/+ brain samples, densitometry analysis of RIG-I signal intensities shows a significant increase in RIG-I protein amount in Rnaset2−/− protein samples (10-fold increase in Rnaset2−/−, p < 0.01). Loading control: β-ACTIN. Mean ± SEM with ** = p < 0.01, Student’s t test; n = 4 per group and time point. WT1-4: Rnaset2+/+, KO1-4: Rnaset2−/−. RIG-I: Retinoic acid-inducible gene I. I Western blot analysis of IRF9 at 28 weeks of age. Blot intensities were measured using densitometry, and results show significantly higher levels of IRF9 protein in Rnaset2−/− samples (ten-fold increase in Rnaset2−/−, p < 0.05). Loading control: β-ACTIN. Mean ± SEM with * = p < 0.05, Student’s t test; n = 3 per group and time point. WT1-3: Rnaset2+/+, KO1-3: Rnaset2−/−. IRF9: interferon regulatory factor 9. J Western blot analysis of RIG-I at 28 weeks of age. Blot densitometry shows significantly upregulated RIG-I protein amounts in Rnaset2−/− brain samples (nine-fold increase in Rnaset2−/−, p < 0.001). Loading control: β-ACTIN. Mean ± SEM with *** = p < 0.001, Student’s t test; n = 3 per group and time point. WT1-3: Rnaset2+/+, KO1-3: Rnaset2−/−. RIG-I: Retinoic acid-inducible gene I.

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