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. 2021 Jan 28;12:640. doi: 10.1038/s41467-020-20839-0

Fig. 1. Cellular stress response under high redox potential activates IFN-I responses.

Fig. 1

a Left, representative images of treated Grx1-roGFP2-NSPC. Right, quantitation for redox potential of Grx1-roGFP2-NSPC following 24 h treatment. Scale bar = 20 µm. Mean ± s.e.m. of 20 cells. PQ: 5 µM of paraquat, NT: mock-treated control, NAC: 5 mM of N-acetylcysteine. b Schema for treatment of NSPC culture. ORP: oxidation/reduction potential. c Representative immunofluorescence analysis (IF) images of TUBB3 on 3 day of differentiation. Scale bar = 50 µm. Percent of TUBB3-positive cells is plotted on the right. Mean ± s.e.m. of three independent experiments. d One-sided GSEA analysis of PQ-upregulated genes showed significant enrichment of ‘Hecker_IFNB1_Targets’ (upper) and ‘Hallmark_IFNA_response’ (lower) gene sets in NSPCs treated with or without PQ. e Heatmap visualization of 108 differentially expressed genes (complete list is available as source data) related to IFN-I response from NSPCs treated with PQ (5 µM) or NAC (5 mM) for 2 days. Top 20 genes are presented on the right. The red represents a positive Z-score (upregulated), and the blue represents a negative one (downregulated). f qRT-PCR results for IFN-1 stimulated genes (ISGs) in NSPCs following 4 days of PQ and NAC treatment. Mean ± s.e.m. of three independent experiments. g IFNβ secretion in the media following 48 h treatment. Mean ± s.e.m. of three independent experiments. h Representative IF (left) and western blot analysis (WB, right) results of NSPCs differentiated following 2 days of IFNβ (40 ng/mL) treatment. Scale bar = 50 µm. i Representative IF images of TUBB3 in NSPCs expressing non-targeted guide RNA (sg-NT) or Ifnar2 targeted guide RNA (sg-Ifnar2) following 3 days of differentiation. Scale bar = 50 µm. Percent of TUBB3-positive cells is plotted on the right. Mean ± s.e.m. of three independent experiments. Statistical significance was determined by one-way ANOVA for a, c, f, and i, and by two-sided unpaired t-test for (g). Experiments for a, c, h, and i were repeated three times independently with similar results and representative images/blots are shown.