Fig 2: Direct effects of glucocorticoids in ependymal glia. (A–I) Zebrafish spinal cord transverse sections. (A–C) Immunostaining of wild-types for Nr3c1 (green) and Gfap (red), plus DAPI staining (blue) with Dex treatment at 24 h post sham injury (A), 24 h (B) and 48 h post SCI (C). Nuclear Nr3c1 (active) localization in Gfap-positive cells around the central canal is stimulated with Dex treatment. (D–F) Immunostaining for EGFP (pan-cytoplasmic) in the SR4G transgenic reporter line (green) and filamentous Gfap (red) at 24 h post sham injury (D) and SCI with no treatment (E) or 24 h post SCI +Dex (F). Nr3c1 signaling is constitutively active in ependymal glia in uninjured controls (D), diminished after SCI (E), and was maintained after SCI + Dex. (G–I) Immunostaining for Sox2 (green), PCNA (red) and Gfap (blue) from 48 h post sham injury (G), 48 h post SCI followed by no treatment (H), or 48 h post SCI +Dex (I). Sox2 immunoreactivity is reduced in ependymal glia and neural precursor cells after Dex treatment. Representative images from 10 spinal cords per time point in each condition. Scale bars, 20 µm (A is the same as B,C; D is the same as E,F; G is the same as H,I). *denotes central canal.

Fig 3: GR DNA-binding mutants are competent for repression at NF-?B GR-tethering sites.U2OS cells were transfected with hGR (WT, C421G or K442E)-expressing or empty EF.CMV.RFP vector (a, left) using Lipofectamine 3000 (ThermoFisher) and treated the next day with vehicle (Veh), 100 nM Dex, 5 ng/ml TNF or TNF+Dex, as indicated. (a) Gene expression of GR-activated IGFBP1 and GILZ (middle) or GR-repressed TNF and IL8 (right) following 2 h treatment was assessed by RT-qPCR with b-Actin as housekeeping control. IGFBP1 (n=5) and GILZ (n=4) fold induction by Dex is expressed over transcript level in veh-treated cells (=1). TNF and IL8 expression (n=3 each) is calculated relative to the transcript level in TNF-treated cells (=1). (b) ChIP with polyclonal antiGR Abs (PA1-511A ThermoFisher) was performed following 2-h Dex (left) or 1-h TNF or TNF+Dex (right) treatment, and GR occupancy was assessed at GREs and NF-?B sites at indicated genomic locations with signals at 28S ribosomal gene as normalization control (n=3). Occupancy is expressed over baseline in veh-treated cells (=1). Error bars are SEM; 2-tailed Student’s t-test.

Fig 4: Association of CREB binding sites with additional transcription factor occupancy at fasting-responsive genes. CREB sites were separated into 3 groups: 285 sites associated with genes up-regulated in fasted state (red), 321 sites associated with genes down-regulated in fasted state (green), 6,018 sites associated with genes that did not change (gray). A-F) Average ChIP-seq profiles around each group of CREB binding sites are shown for CREB (A), CEBPB (B), NR3C1/GR (C), FOXA2 (D), PPARA (E), and CTCF (F). Corresponding distributions of peak heights are shown as box plots in Additional file 1: Figure S7 and tabulated in Additional file 3. G) Percentage of sites in each group containing the indicated motifs within +/- 250 bp of peak center. *p-value < 0.001 by Fisher’s exact test compared to gray bar.

Fig 5: Glucocorticoids inhibit functional recovery following SCI. Locomotor recovery score of wild-type or nr3c1 targeting TALEN-injected zebrafish that received no treatment or Dex measured at 6, 24, 48, 72, and 120 h post sham injury or SCI. Locomotor responses were quantified from uninjured controls with no treatment controls (blue line), Dex (red line), and nr3c1 TALEN injections (black line), or from SCI with no treatment (green line), Dex (purple line), and Dex with nr3c1 TALEN injections combined (yellow line). Data show the mean ± SEM; n = 10 per time point in each condition (*p < 0.05; **p < 0.01 [compared to uninjured controls], and #p < 0.01 [compared to both uninjured controls and SCI no treatment]). * and # color coded to match corresponding data points.