Journal: Molecular Therapy

Article Title: siRNA for REST ameliorates symptoms in ALS mice and serum REST predicts disease prognosis and survival in ALS patients

doi: 10.1016/j.ymthe.2025.10.039

Figure Lengend Snippet: Serum levels and prognostic value of REST protein in ALS patients (A) ELISA assay results showing REST protein levels in the serum of ALS patients ( n = 56), compared with healthy controls (Ctrl) ( n = 23). ∗∗ p < 0.01 vs. control by Student’s t test. (B) Kaplan-Meier analysis of tracheostomy-free survival in ALS patients stratified according to the median serum REST protein levels. Patients with REST levels above the median, represented in red, exhibit a significantly worse prognosis than patients with levels below the median, shown in light green. (C and D) Univariate and multivariate Cox proportional hazard models assessing the prognostic value of increased serum REST levels, adjusted for bulbar onset type, sex, progression rate, and age at serum sampling. Whiskers represent the 95% confidence interval (CI). ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: Serum REST level was quantified by a human-specific ELISA kit specific for REST (Cusabio, American Research Products, Waltham, MA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Sampling

Journal: Endocrinology

Article Title: PROX1 Promotes Secretory Granule Formation in Medullary Thyroid Cancer Cells.

doi: 10.1210/en.2015-1973

Figure Lengend Snippet: Figure 5. Effect of PROX1 on known CHGA transcription-regulatory factors. A, A schematic diagram of the CHGA promoter region and the putative PROX1 binding site. CRE, cAMP-responsive element: ex, exon; RE1, repressor element 1; TSS, transcription start site. ChIP-PCR target indicates the region amplified in the chromatin immunoprecipitation- based PCR assay. B, Semiquantitative RT-PCR analysis of PROX1 and CHGA, REST mRNA expression, and Western blot analysis of REST, CREB, phosphorylated CREB (CREB-P), and -actin expression in PROX1-inducible KTC1 and TTA1 cells in the absence (0 h) or presense (12, 24, and 48 h) of the inducer Dox. Data are shown as means SD. C, ChIP-PCR assay of CREB and REST binding to the CHGA gene. Cell lysates of Dox-treated or untreated FLAG-tagged PROX1-transfected KTC1 cells (KTC1-PROX1) were immunoprecipitated by using anti- CREB or anti-PROX1 antibody or normal rabbit IgG. Coprecipitation of gene fragments of CHGA containing CRE or RE1 sequence (CHGA[481 to 367] or CHGA[272 to 808], respectively) were assayed using PCR. CREB, rabbit anti-CREB antibody; IgG, normal rabbit IgG; IP, immunoprecipitation; REST, rabbit anti-REST antibody. The experiment was conducted in triplicate. Data are shown as means SD.

Article Snippet: Briefly, the chromatin solution was incubated with 1 g or recommended amount of mouse anti-FLAG antibody (Sigma-Aldrich), rabbit anti-CREB (Cell Signaling Technology), rabbit anti-REST antibody (Bethyl Laboratories, Inc), normal mouse IgG (Santa Cruz Biotechnology), or normal rabbit IgG (Santa Cruz Biotechnology), and the antibody/chromatin mixtures were then precipitated with Protein G beads.

Techniques: Binding Assay, Amplification, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Immunoprecipitation, Sequencing

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: PLoS Genetics

Article Title: The Transcription Factor REST Is Lost in Aggressive Breast Cancer

doi: 10.1371/journal.pgen.1000979

Figure Lengend Snippet: (A) MCF7 breast cancer cells were infected with lentivirus bearing either non-targeting control shRNA or REST targeting shRNA. REST knockdown was verified by western blot (Millipore antibody). Beta actin expression in shown as a loading control. (B) Paraffin-embedded MCF7 sections were stained with antibody to the REST C-terminus (Sigma) (stained brown), with hemetoxylin nuclear stain (stained blue). Control shRNA expressing cells show strong nuclear REST staining, while REST shRNA expressing cells show little to no nuclear REST staining.

Article Snippet: To determine the frequency of REST protein loss in breast cancer, we developed an immunohistochemical (IHC) screen using an antibody directed to the C-terminus of REST (Atlas Antibodies, Stockholm).

Techniques: Infection, Control, shRNA, Knockdown, Western Blot, Expressing, Staining

Journal: PLoS Genetics

Article Title: The Transcription Factor REST Is Lost in Aggressive Breast Cancer

doi: 10.1371/journal.pgen.1000979

Figure Lengend Snippet: (A,B) Paraffin embedded breast tumor sections were immunohistochemically labeled with an antibody to the C-terminus of REST. Inset image is enlarged 2.5× to show detail. (A) Strong nuclear REST labeling in the majority of tumor cells (brown signal). (B) Weak or absent labeling for REST in REST–less tumor. (C,D) Paraffin embedded breast tumor sections were stained for the protein product of the REST target gene chromogranin-A ( CHGA ). (C) No CGA labeling detected in RESTfl tumor (blue hematoxylin stain highlights nuclei of carcinoma and stroma cells). (D) Moderate to strong labeling for CGA in a subset of carcinoma cells. The brown label (CGA) does not co-localize with the nuclear hematoxylin counterstain, suggesting a non-nuclear location. REST–less tumors were significantly enriched in chromogranin-A staining (D, p<0.001).

Article Snippet: To determine the frequency of REST protein loss in breast cancer, we developed an immunohistochemical (IHC) screen using an antibody directed to the C-terminus of REST (Atlas Antibodies, Stockholm).

Techniques: Labeling, Staining

Journal: PLoS Genetics

Article Title: The Transcription Factor REST Is Lost in Aggressive Breast Cancer

doi: 10.1371/journal.pgen.1000979

Figure Lengend Snippet: Patient outcome and tumor data corresponding to 182 paraffin embedded breast tumors stained with antibody to the REST C-terminus.

Article Snippet: To determine the frequency of REST protein loss in breast cancer, we developed an immunohistochemical (IHC) screen using an antibody directed to the C-terminus of REST (Atlas Antibodies, Stockholm).

Techniques: Staining

Journal: Antioxidants (Basel, Switzerland)

Article Title: Hydrogen Peroxide-Induced Re-Expression of Repressor Element 1-Silencing Transcription Factor Contributes to Cardiac Vagal Dysfunction in Type 2 Diabetes Mellitus.

doi: 10.3390/antiox14050588

Figure Lengend Snippet: Figure 3. Effect of REST shRNA transfection on N-type Ca2+ channel (Cav2.2-α) protein expression in AVG neurons from T2DM rats. (A) Raw and quantitative data representing the REST protein levels in AVG neurons from all groups of rats, analyzed by reverse-phase protein microarray. (B) Raw and quantitative data representing Cav2.2-α protein levels in the AVG neurons from all groups of rats. Black dots represent each individual data point. N = 12 measurements from 6 rats/group. Data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

Article Snippet: Under the microscope, 2 μL of saline, adenoviral catalase gene (Ad.CAT, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), adenoviral vector control (Ad.Empty, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), rat lentiviral REST shRNA (Lenti.REST shRNA, 29mer target-specific shRNA designed against multiple slice variants based on NM_031788 and NM_031788.1, 1 × 107 TU/mL, CAT#: TL711581V, OriGene Technologies, Inc., Rockville, MD, USA), or lentiviral scrambled shRNA (Lenti.scrambled shRNA, lenti particles carrying a 29mer scrambled shRNA sequence cassette, 1 × 107 TU/mL, CAT#: TR30021V, OriGene Technologies, Inc., Rockville, MD, USA) was microinjected into the AVG by a glass micropipette connected to a WPI Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA).

Techniques: shRNA, Transfection, Expressing, Microarray

Journal: Antioxidants (Basel, Switzerland)

Article Title: Hydrogen Peroxide-Induced Re-Expression of Repressor Element 1-Silencing Transcription Factor Contributes to Cardiac Vagal Dysfunction in Type 2 Diabetes Mellitus.

doi: 10.3390/antiox14050588

Figure Lengend Snippet: Figure 4. Effect of REST shRNA transfection on N-type Ca2+ currents in AVG neurons from T2DM rats. (A) Raw data for Ca2+ currents from sham, T2DM, and T2DM + lenti.REST shRNA rats. (B) Quantitative data for total Ca2+ currents and other types of Ca2+ currents (cells treated with ω-conotoxin GVIA, a specific N-type Ca2+ channel blocker) recorded under the test pulse at 0 mV in AVG neurons from all groups of rats. (C) Quantitative data for N-type Ca2+ currents analyzed from the original recording in AVG neurons from all groups of rats. Subtracting Ca2+ currents under treatment of ω-conotoxin GVIA from total Ca2+ currents in the original recording was used to obtain N-type Ca2+ currents. Black dots represent each individual data point. N = 6 neurons from 4 rats/group; data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

Article Snippet: Under the microscope, 2 μL of saline, adenoviral catalase gene (Ad.CAT, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), adenoviral vector control (Ad.Empty, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), rat lentiviral REST shRNA (Lenti.REST shRNA, 29mer target-specific shRNA designed against multiple slice variants based on NM_031788 and NM_031788.1, 1 × 107 TU/mL, CAT#: TL711581V, OriGene Technologies, Inc., Rockville, MD, USA), or lentiviral scrambled shRNA (Lenti.scrambled shRNA, lenti particles carrying a 29mer scrambled shRNA sequence cassette, 1 × 107 TU/mL, CAT#: TR30021V, OriGene Technologies, Inc., Rockville, MD, USA) was microinjected into the AVG by a glass micropipette connected to a WPI Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA).

Techniques: shRNA, Transfection

Journal: Antioxidants (Basel, Switzerland)

Article Title: Hydrogen Peroxide-Induced Re-Expression of Repressor Element 1-Silencing Transcription Factor Contributes to Cardiac Vagal Dysfunction in Type 2 Diabetes Mellitus.

doi: 10.3390/antiox14050588

Figure Lengend Snippet: Figure 5. Effect of REST shRNA transfection on neuronal excitability in AVG neurons from T2DM rats. Raw (A) and quantitative data (B) for action potentials during a 1 s current clamp with a current injection of 100 pA in AVG neurons from all groups of rats. Black dots represent each individual data point. N = 6 neurons from 4 rats/group; data are means ± SEM. One-way ANOVA with post-hoc Bonferroni test was used to assess statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

Article Snippet: Under the microscope, 2 μL of saline, adenoviral catalase gene (Ad.CAT, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), adenoviral vector control (Ad.Empty, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), rat lentiviral REST shRNA (Lenti.REST shRNA, 29mer target-specific shRNA designed against multiple slice variants based on NM_031788 and NM_031788.1, 1 × 107 TU/mL, CAT#: TL711581V, OriGene Technologies, Inc., Rockville, MD, USA), or lentiviral scrambled shRNA (Lenti.scrambled shRNA, lenti particles carrying a 29mer scrambled shRNA sequence cassette, 1 × 107 TU/mL, CAT#: TR30021V, OriGene Technologies, Inc., Rockville, MD, USA) was microinjected into the AVG by a glass micropipette connected to a WPI Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA).

Techniques: shRNA, Transfection, Injection

Journal: Antioxidants (Basel, Switzerland)

Article Title: Hydrogen Peroxide-Induced Re-Expression of Repressor Element 1-Silencing Transcription Factor Contributes to Cardiac Vagal Dysfunction in Type 2 Diabetes Mellitus.

doi: 10.3390/antiox14050588

Figure Lengend Snippet: Figure 6. Effect of REST shRNA on vagal control of the ventricular function in T2DM. Left vagal efferent nerve stimulation (VNS)-induced changes of the left ventricular systolic pressure (LVSP, panel A) and the maximum rate of increase of left ventricular pressure (LV dP/dtmax, panel B) represented the vagal control of the ventricular function. N = 6 rats/group; data are means ± SEM. Two-way repeated measures ANOVA with post-hoc Bonferroni was used to test statistical significance. * p < 0.05 vs. sham; # p < 0.05 vs. T2DM.

Article Snippet: Under the microscope, 2 μL of saline, adenoviral catalase gene (Ad.CAT, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), adenoviral vector control (Ad.Empty, 1 × 1010 pfu/mL, University of Iowa, Iowa City, IA, USA), rat lentiviral REST shRNA (Lenti.REST shRNA, 29mer target-specific shRNA designed against multiple slice variants based on NM_031788 and NM_031788.1, 1 × 107 TU/mL, CAT#: TL711581V, OriGene Technologies, Inc., Rockville, MD, USA), or lentiviral scrambled shRNA (Lenti.scrambled shRNA, lenti particles carrying a 29mer scrambled shRNA sequence cassette, 1 × 107 TU/mL, CAT#: TR30021V, OriGene Technologies, Inc., Rockville, MD, USA) was microinjected into the AVG by a glass micropipette connected to a WPI Nanoliter 2000 microinjector (World Precision Instruments, Sarasota, FL, USA).

Techniques: shRNA, Control