Article Figures & Data
Figures
- Fig. 1.
C-DIM5 alone and MPTP/IFNγ/TNFα-induced mRNA expression in primary mixed glia over 24 hours. (A) Flow cytometry scatter plots show the percentage of Cd11b (microglia, 12%) or GLAST-positive (astrocytes, 78.8%) cells in mixed glial cultures. (B) C-DIM5 alone increases Nur77 mRNA at 4 hours and MPTP/IFNγ/TNFα mildly activates Nur77; both synergistically show an increase. (C) C-DIM5 alone does not increase Nurr1. (D) Time course over 24 hours demonstrates the most inflammatory gene expression at 4 hours, whereas C-DIM5 does not. (E) C-DIM5 suppresses inflammatory gene expression at 4 hours. Data depicted as ±S.E.M. (n = 3–14); mRNA fold change; internal control (β-actin or HPRT). Statistical significance shown as mean compared with control except for those depicted in 2E, where statistical significance is compared with MPTP/TNF/IFN. *P < 0.05; **P < 0.01; ****P < 0.0001.
- Fig. 2.
C-DIM5 inhibits NF-κB activity in HEK NF-κB-GFP/luciferase reporter cells upon hNR4A1-FLAG (Nur77) overexpression through a nuclear specific mechanism exhibited in primary astrocytes. (A) Western blot and representative images demonstrating increased (arrows) Nur77 and FLAG in HEK dual-reporter cells. (B) Representative images demonstrating increased nuclear Nur77 upon overexpression in the presence of C-DIM5 after TNF treatment in HEK cells. (C and D) Quantitative graphs demonstrate decreased NF-κB activity via luciferase and GFP expression. (E) A time-course graph demonstrating increased nuclear p65 expression under the influence of MPTP and IFNγ/TNFα at 30 minutes in primary astrocytes. Additionally, C-DIM5 does not suppress p65 translocation. (F) Representative images of p65 translocation in astrocytes under basal conditions and/or stimulation with MPTP (10 μM) and TNF-α (10 pg/μl)/IFN-γ (1 ng/μl) in the absence or presence of 1 μM C-DIM5. Data depicted as ±S.E.M *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 n = 100–200 cells per group from three biologic replicates across three independent experiments.
- Fig. 3.
C-DIM5 prevents Nur77 (NR4A1) from translocating to the cytoplasm and shuttles Nurr1 (NR4A2) to the nucleus under inflammatory stimuli. (A) Representative images of Nur77 translocation in astrocytes under control (saline) conditions and/or stimulation with MPTP/IFNγ/TNFα in the absence or presence of C-DIM5. (B) A quantitative figure demonstrating nuclear Nur77 expression at time point 30 minutes upon inflammatory stimuli and/or C-DIM5 treatment. (C) Representative images of Nurr1 translocation in astrocytes under control (saline) conditions and/or stimulation with MPTP/IFNγ/TNFα in the absence or presence of C-DIM5. (D) A quantitative figure demonstrating nuclear Nurr1 expression at time point 30 minutes upon inflammatory stimuli and/or C-DIM5 treatment or DMSO-vehicle control treatment. Data depicted as ±S.E.M *P < 0.05; **P < 0.01; ****P < 0.0001 n = 100–200 cells per image field per group for n = 3 across three independent experiments.
- Fig. 4.
RNAi targeted toward Nur77 and Nurr1 effectively knocks down Nur77 and Nurr1 mRNA expression in primary pure astrocytes, respectively. (A) Nur77 is silenced alone at 48 hours, which selectively increases expression of NR4A2/Nurr1 without affecting expression of NR4A3/NOR1. (B) Nurr1 is silenced alone, which selectively increases expression of NR4A1/Nur77 without affecting expression of NR4A3/NOR1 (C) Double knockdown of both Nur77 and Nurr1 effectively suppresses Nur77 and Nurr1, respectively. (D) Transfecting primary mixed glia effectively removes microglia yielding pure GLAST-positive primary astrocytes, as demonstrated by flow cytometry. (E) Twenty-four hour treatment with C-DIM5 alone or with MPTP-cytokines shows increased Nur77 protein compared with less Nurr1 protein. C-DIM5 effectively increases Nur77 at 24 hours versus 8 hours, whereas Nurr1 expression is expressed more at 8 hours. (F) Double knockdown prevents Nos2, Il1β, Il6, and Tnfα suppression by C-DIM5 with MPTP-cytokines. Data depicted as ±S.E.M. (n = 4–22/group); mRNA fold change; internal control (β-actin or HPRT). Statistical significance shown as mean compared with control. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
- Fig. 5.
ChIP-seq analysis of p65-bound genes in C-DIM5-treated MPTP-exposed astrocytes. C-DIM5 largely restores NF-κB-mediated inflammatory and apoptosis gene expression back to control levels as measured by a qPCR array study. (A) Venn diagram depicting unique and overlapping genes bound to p65 in C-DIM5 and vehicle (DMSO)-treated MPTP exposed astrocytes. (B) Bar plot for the gene ontology analysis of biologic processes found to be statistically unique in the C-DIM5-treated MPT-exposed astrocytes. –log(P value) used to rank the degree of enrichment of the top 21 genes. The number next to each bar represents the number of associated genes corresponding to C-DIM5–treated MPTP-exposed astrocytes. (C) Genome browser view of distribution of the ChIP-seq reads of represented genes in both C-DIM5- and DMSO-treated MPTP-exposed astrocytes. (D) A cluster gram and heat map demonstrate gene pattern expression similarities among treatment groups, MPTP/IFNγ/TNFα + C-DIM5 clusters with control (saline) levels; MPTP/IFNγ/TNFα clusters alone. (E) MPTP/IFNγ/TNFα increases gene expression from control, whereas C-DIM5 decreases MPTP/IFNγ/TNFα -induced gene expression. MPTP/IFNγ/TNFα + C-DIM5 has minimal changes in gene expression compared with control. (n = 4/group in array studies).
- Fig. 6.
Small molecule docking of C-DIM5. (A) Predicted binding orientation of C-DIM5 (shown in yellow) at the ligand-binding site of NR4A1 (shown in purple). (B) Inset depicts the specific residues of the calculated binding site in NR4A1 (gray) and their predicted interactions with C-DIM5 as dashed lines (pink=hydrophobic/π-alkyl, purple=π-σ, cyan=π-cation/π-anion, and green=hydrogen bonds). (C) Bond types shown for predicted binding interactions between C-DIM5 and NR4A1. (D) Predicted binding orientation of C-DIM5 (shown in yellow) at the coactivator-binding site of NR4A2 (shown in blue). (E) Inset depicts the specific residues of the calculated binding site in NR4A2 (gray) and their predicted interactions with C-DIM5, with (D) bond types shown for predicted binding interactions between C-DIM5 and NR4A2.
Tables
- TABLE 1
Primer table.
Primer sequences of measured genes in qPCR experiments separate from the SABiosciences NF-κB-targeted gene array.
Gene Accession No. Primer Sequence (5′-3′) Length NOS2 NM_010927.3 For: TCA CGC TTG GGT CTT GTT bp Rev: CAG GTC ACT TTG GTA GGA TTT 149 TNFα NM_013693.3 For: CTT GCC TGA TTC TTG CTT CTG 140 Rev: GCC ACC ACT TGC TCC TAC IL-1β NM_008361.3 For: GCA GCA GCA CAT CAA CAA G 90 Rev: CAC GGG AAA GAC ACA GGT AG NURR1/NR4A2 NM_001139509.1 For: GTG TTC AGG CGC AGT ATG G 153 Rev: TGG CAG TAA TTT CAG TGT TGG T CCL2 NM_011331.2 For: TTAAAAACCTGGATCGGAACCAA 121 Rev: GCATTAGCTTCAGATTTACGGGT CCL5 NM_013653.3 For: GCT GCT TTG CCT ACC TCT CC 104 Rev: TCG AGT GAC AAA CAC GAC TGC IL-6 NM_031168.1 For:CTG CAA GAG ACT TCC ATC CAG 131 Rev:AGT GGT ATA GAC AGG TCT GTT GG β-ACTIN NM_007393.3 For: GCT GTG CTA TGT TGC TCT AG 117 Rev: CGC TCG TTG CCA ATA GTG HPRT NM_013556.2 For: TCA GTC AAC GGG GGA CAT AAA 142 Rev: GGG GCT GTA CTG CTT AAC CAG NOR1/NR4A3 XM_006537657.3 For: TGC GTG CAA GCC CAG TAT AG 60 Rev: ATA AGT CTG CGT GGC GTA AGT NUR77/NR4A1 NM_010444.2 For: TTG GGG GAG TGT GCT AGA AG 202 Rev: GTA GGC TTG CCG AAC TCA AG - TABLE 2
Reduction in secreted cytokines by C-DIM5
Inflammatory cytokines secreted upon MPTP/IFNγ/TNFα treatment are significantly reduced upon addition of C-DIM5.
Group Secreted Cytokine Concentration (pg/ml) TARC IL2 IL6 IL-12p70 CCL2 MIP-1a CCL5 Control — 1.9 ± 0.65* -** 13.2 ± 0.67** -* 5.7 ± 0.52** 9.6 ± 0.47* MPTP/TNF/IFN 3.1 ± 0.24 84.6 ± 1.13 7.4 ± 0.33 750.4 ± 7.58 4.1 ± 1.09 1916 ± 153.55 18.4 ± 0.80 MPTP/TNF/IFN + C-DIM5 2.6 ± 0.15 55.1 ± 2.53** 2.4 ± 0.27** 279.5 ± 5.74** —* 111.2 ± 3.08** 16.6 ± 1.46* —, Result lower than limit of detection.
↵* P < 0.01; **P < 0.001 Statistical significance from MPTP/TNF/IFN group (n = 4/group).
Data Supplement
- Supplement Figure 1 - PDB -
Crystal structure coordinates. Protein data bank file for the crystal structure coordinates for the NR4A1 ligand binding domains (PDB ID: 1YJE) (Data Supplement Figure 1) downloaded from the protein data bank (pdb format) used to assist in predicted binding orientation of C-DIM5.
- Supplemental Figure 2 - PDB -
Crystal structure coordinates. Protein data bank file for the crystal structure coordinates for the NR4A2 ligand-binding domain (PDB ID: 1OVL) (Data Supplement Figure 2) downloaded from the protein data bank (pdb format) used to assist in predicted binding orientation of C-DIM5.
- Supplement Figure 1 - PDB -
