Figure 4.
Characterization of chemogenetic astrocyte reactivity and discovery of downstream intracellular and extracellular signaling mechanisms. (A and B) Subset of inflammatory transcripts upregulated (purple) in day 14 Astrostim cells treated with CNO for 2 h (A) or 2 d (B), compared with nontreated control cells (gray; n = 3 each), as detected by RNAseq; see Data S4. (C) Top 5 KEGG pathways significantly upregulated in 2-h CNO treatment of Astrostim cells, shown as a simplified connectome map (CNet plot). Pathways are shown as hubs (gray, containing KEGG IDs and number of DEGs in pathway) with associated genes (purple). See Fig. S5 B and Data S4. For all pathways, Padj ≤ 0.0001. KS, Kaposi sarcoma. (D) CNO and IL-1α+TNF-α (10 ng/ml) treatments increased NF-κB nuclear translocation compared with controls. Shown are NF-κB (white) and DAPI (magenta) signals with insets of single cells (top left) and nuclear NF-κB confined to DAPI mask regions (top right), as well as nuclear NF-κB signal intensities normalized to controls (bottom; n = 3 each). (E) Gene expression of the inflammatory marker CCL2 significantly decreased with 2-h pretreatment of 20 µM Dex preceding 2-h and 2-d CNO treatment of Astrostim cells (normalized to non–Dex-treated controls; n = 5 each). (F) Gene expression of the inflammatory marker CCL2 decreased with 2-h pretreatment of 1 µM IκB inhibitor ACHP preceding 2-h and 2-d CNO treatment of Astrostim cells (normalized to non–ACHP treated controls; n = 4 each). (G) Quantification of CCL2 protein in cell lysate (left) or conditioned media (right) was quantified by ELISA. As expected, CNO treatment significantly increased CCL2 protein levels from non–CNO treated controls. Application of ACHP or Dex to Astrostim cells preceding CNO treatment significantly decreased CCL2 protein levels (n = 3 each). Asterisks under bars indicate a significant increase in CCL2 levels with CNO treatment, compared with the control; asterisks above bars indicate a significant decrease in CCL2 levels with drug pretreatments, compared with CNO treatment alone. All scale bars: 100 µm; inset of D: 10 µm. Results are shown as mean ± SEM. For D–F, significance was determined using two-tailed unpaired t tests. For G, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test, with *, P or Padj ≤ 0.05; **, P or Padj ≤ 0.01; ***, P or Padj ≤ 0.001; ****, P or Padj ≤ 0.0001.

Characterization of chemogenetic astrocyte reactivity and discovery of downstream intracellular and extracellular signaling mechanisms. (A and B) Subset of inflammatory transcripts upregulated (purple) in day 14 Astrostim cells treated with CNO for 2 h (A) or 2 d (B), compared with nontreated control cells (gray; n = 3 each), as detected by RNAseq; see Data S4. (C) Top 5 KEGG pathways significantly upregulated in 2-h CNO treatment of Astrostim cells, shown as a simplified connectome map (CNet plot). Pathways are shown as hubs (gray, containing KEGG IDs and number of DEGs in pathway) with associated genes (purple). See Fig. S5 B and Data S4. For all pathways, Padj ≤ 0.0001. KS, Kaposi sarcoma. (D) CNO and IL-1α+TNF-α (10 ng/ml) treatments increased NF-κB nuclear translocation compared with controls. Shown are NF-κB (white) and DAPI (magenta) signals with insets of single cells (top left) and nuclear NF-κB confined to DAPI mask regions (top right), as well as nuclear NF-κB signal intensities normalized to controls (bottom; n = 3 each). (E) Gene expression of the inflammatory marker CCL2 significantly decreased with 2-h pretreatment of 20 µM Dex preceding 2-h and 2-d CNO treatment of Astrostim cells (normalized to non–Dex-treated controls; n = 5 each). (F) Gene expression of the inflammatory marker CCL2 decreased with 2-h pretreatment of 1 µM IκB inhibitor ACHP preceding 2-h and 2-d CNO treatment of Astrostim cells (normalized to non–ACHP treated controls; n = 4 each). (G) Quantification of CCL2 protein in cell lysate (left) or conditioned media (right) was quantified by ELISA. As expected, CNO treatment significantly increased CCL2 protein levels from non–CNO treated controls. Application of ACHP or Dex to Astrostim cells preceding CNO treatment significantly decreased CCL2 protein levels (n = 3 each). Asterisks under bars indicate a significant increase in CCL2 levels with CNO treatment, compared with the control; asterisks above bars indicate a significant decrease in CCL2 levels with drug pretreatments, compared with CNO treatment alone. All scale bars: 100 µm; inset of D: 10 µm. Results are shown as mean ± SEM. For D–F, significance was determined using two-tailed unpaired t tests. For G, significance was determined using a one-way ANOVA followed by Tukey’s multiple comparison test, with *, P or Padj ≤ 0.05; **, P or Padj ≤ 0.01; ***, P or Padj ≤ 0.001; ****, P or Padj ≤ 0.0001.

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