Journal: Scientific Reports

Article Title: A selective inhibitor of histone deacetylase 3 prevents cognitive deficits and suppresses striatal CAG repeat expansions in Huntington’s disease mice

doi: 10.1038/s41598-017-05125-2

Figure Lengend Snippet: RGFP966 treatment restores gene expression in the hippocampus of HD mice. (a) Schematic time line of acute treatment. ( b) Quantitative RT-PCR analysis of memory-related genes in the hippocampus of vehicle and RGFP966 acutely treated Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) mice. Histogram represents relative mRNA abundance of Arc, Nr4a2, Egr1 and c-Fos . Levels of mRNA were normalized to 18S and Actinβ. * p < 0.05 compared to vehicle-treated WT mice by two-way ANOVA with Bonferroni post-hoc analysis. (c) Representative images (high magnification) showing Arc immunostaining in the hippocampal dentate gyrus of acutely treated Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) mice. Histogram shows quantification of the average number of Arc-positive neurons in the dentate gyrus. * p < 0.05 compared to vehicle-treated WT mice by two-way ANOVA with Bonferroni post-hoc analysis. (d) Representative images with magnification insets of the hippocampal CA1 region showing Egr1 immunostaining in acutely treated Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) mice. Histogram shows quantification of the average intensity of Egr1 immunoreactivity in the CA1 or the CA3 region. Data represent the mean ± SEM (n = 5–8 animals per group).

Article Snippet: Antibodies used for immunoblot analysis were: Acetyl-histone H3 (Lys9) (1:1000, Cell Signaling Technology), Histone H3 (1:1000, Cell Signaling Technology), HDAC3 (1:1000; Abcam), Arc (1:500; Santa Cruz Biotechnology), Egr1 (1:1000; Cell Signaling Technology), c-Fos (1:1000, Santa Cruz Biotechnology), DARPP-32 (1:1000; BD Bioscience), PDE10A (1:1000; Abcam), A 2A R (1:500; Santa Cruz Biotechnology), 1C2 (1:1000, Millipore), α-Tubulin (1:50,000; Sigma-Aldrich) and Actin (1:20,000; MP Biochemicals).

Techniques: Expressing, Quantitative RT-PCR, Immunostaining

Journal: Scientific Reports

Article Title: A selective inhibitor of histone deacetylase 3 prevents cognitive deficits and suppresses striatal CAG repeat expansions in Huntington’s disease mice

doi: 10.1038/s41598-017-05125-2

Figure Lengend Snippet: RGFP966 treatment induces Arc but not Egr1 or c-Fos protein levels in primary hippocampal cultures. (a ) Representative immunoblots showing histone H3 acetylation levels at position lysine 9 (AcH3K9) with actin as loading control and (b) Arc, Egr1 and c-Fos protein levels normalized to actin levels in DMSO and RGFP966 treated primary hippocampal cultures obtained from Hdh Q7/Q7 (WT) and Hdh Q7/Q111 (KI) embryos. The blots in (a and b) are cropped; full-length images are provided in Supplementary Figure . ** p < 0.01 compared to DMSO-treated cultures by two-way ANOVA with Bonferroni post-hoc analysis. Data represent the mean ± SEM (n = 4–5 cultures per group).

Article Snippet: Antibodies used for immunoblot analysis were: Acetyl-histone H3 (Lys9) (1:1000, Cell Signaling Technology), Histone H3 (1:1000, Cell Signaling Technology), HDAC3 (1:1000; Abcam), Arc (1:500; Santa Cruz Biotechnology), Egr1 (1:1000; Cell Signaling Technology), c-Fos (1:1000, Santa Cruz Biotechnology), DARPP-32 (1:1000; BD Bioscience), PDE10A (1:1000; Abcam), A 2A R (1:500; Santa Cruz Biotechnology), 1C2 (1:1000, Millipore), α-Tubulin (1:50,000; Sigma-Aldrich) and Actin (1:20,000; MP Biochemicals).

Techniques: Western Blot

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 2. Sp1 activates and is essen- tial for full pgp2/mdr1b promoter ac- tivity. A, radiolabeled pgp2GC or mtpgp- 2GC oligonucleotide was incubated with (1) and without (2) affinity-purified Sp1 (one footprinting unit) and analyzed by EMSA. The last lane with mtpgp2GC con- tains 5 footprinting units of Sp1. B, the activity of 2250WT promoter was com- pared with the 2250MT pgp2/mdr1b mu- tant promoter that contained the identi- cal mutations as were found in the mtpgp2GC oligonucleotide. 10 mg of each of these plasmids were transiently trans- fected into H35 cells and luciferase activ- ity measured. Results are expressed as activity relative to the 2250WT promoter (100%). C, Drosophila Schneider cells were transiently transfected with 1 mg of the 2250WT or 2250MT plasmid along with indicated amounts of pPacSp1 (wild- type Sp1 expression plasmid) or N539 (a COOH-terminal Sp1 deletion mutant) and luciferase activity measured. Values in B and C are the average of three sepa- rate transfection experiments each per- formed in duplicate to quadruplicate. The error bars represent the standard error of the mean. Results are expressed as fold- activation relative to 2250WT (1.0) or 2250MT (1.0) co-transfected with empty vector.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Incubation, Affinity Purification, Footprinting, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Activation Assay

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 3. Pgp and Sp1 levels and Sp1 DNA binding in H35 clones stably ex- pressing Sp1 (Sp1–9, 10, 18, or 20) or Neo vector (pc2). A, analysis of Sp1: 50 mg of nuclear extract protein from the in- dicated cell lines was analyzed by immu- noblot with anti-Sp1 IgG. B, a radiola- beled consensus Sp1 oligonucleotide was incubated with nuclear extracts prepared from H35 clones stably expressing Sp1 or pcDNA vector and analyzed by EMSA. C, analysis of Pgp: 35 mg of total cell lysate protein from H35 clones was analyzed by immunoblot.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Binding Assay, Clone Assay, Stable Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 4. Pgp2/mdr1b binds authentic Egr-1 and Egr-1 in H35 Cells. A, 32P end-labeled pgp2GC oligonucleotide was incubated with H35 nuclear extract in the presence or absence (NC, no competitor) of excess unlabeled pgp2GC, Sp1, Egr, or Oct-1 oligonucleotide (see “Ex- perimental Procedures” for amounts) and analyzed by EMSA. B, 32P- labeled pgp2GC or Egr consensus oligonucleotides were incubated with in vitro translated Egr1 in the presence or absence (NC, no competitor) of various competitor oligonucleotides. The molar excess of competitors was as follows: Oct-1 (200-fold), pgp2GC (100-fold), EgrWT (100-fold), and EgrMT (200-fold).

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Labeling, Incubation, In Vitro

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 6. Competitive displacement of Sp1 binding to the 236 GC-rich region in the Pgp2/mdr1b promoter by Egr-1. EMSA was performed on 32P end-labeled pgp2GC oligonucleotide (left panel) incu- bated in the presence of a constant amount of Sp1 (1 footprinting unit/incubation) and increasing amounts of bacterially expressed Egr- 1-GST fusion protein, or (right panel) incubated with a fixed amount of recombinant Egr-1-GST (2 ml/reaction) and increasing amounts of re- combinant Sp1 (1–10 footprinting units).

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Binding Assay, Labeling, Footprinting, Incubation, Recombinant

Journal: The Journal of biological chemistry

Article Title: Sp1 and egr-1 have opposing effects on the regulation of the rat Pgp2/mdr1b gene.

doi: 10.1074/jbc.274.5.3199

Figure Lengend Snippet: FIG. 8. Enforced expression of Egr-1 in H35 hepatoma cells affects mdr1 expression. H35 cells were transfected with either the Egr-1 expression vector or pcDNA3, placed under G418 selection and Egr-Neo and Neo clones isolated, expanded and characterized by West- ern analysis for Egr-1 and Sp1 expression. The loading control was a nonspecific band that appears equivalent in all samples.

Article Snippet: Polyclonal antibodies to Egr-1 and Sp1 were purchased from Santa Cruz Biotech, and the mdr1 (Ab-1) and actin antibodies were purchased from Calbiochem (La Jolla, CA).

Techniques: Expressing, Transfection, Plasmid Preparation, Selection, Clone Assay, Isolation, Control