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: The Journal of Biological Chemistry

Article Title: HKDC1 promotes colorectal cancer progression by regulating RCOR1 expression to activate the Wnt/β-catenin pathway, enhancing proliferation, migration, and epithelial-mesenchymal transition

doi: 10.1016/j.jbc.2025.108478

Figure Lengend Snippet: HKDC1 regulates RCOR1 to promote the proliferation and migration of CRC cells . A , survival analysis of RCOR1. B , co-immunoprecipitation (Co-IP) analysis of HKDC1 and RCOR1 interaction. C , WB analysis of the regulatory relationship between HKDC1 and RCOR1. D and E , EdU and colony formation assays comparing the proliferation capacity of various CRC cell groups. F and G , Transwell and wound healing assays were conducted to compare the migration capacities of various CRC cell groups. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime Biotechnology) for 2 h and incubated overnight with primary antibodies: HKDC1 (1:1000, Abcam), RCOR1 (1:1000, Proteintech), Bax (1:2000, Proteintech), Bcl-2 (1:2500, Proteintech), CDK4 (1:1000, Proteintech), CDK6 (1:2000, Proteintech), Cyclin D1 (1:5000, Proteintech), β-catenin(1:5000, Proteintech), TCF4 (1:2000, Proteintech), c-Myc(1:2000, Proteintech), N-cadherin (1:5000, Proteintech), E-cadherin (1:50,000, Proteintech), and vimentin (1:1000, Proteintech).

Techniques: Migration, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: The Journal of Biological Chemistry

Article Title: HKDC1 promotes colorectal cancer progression by regulating RCOR1 expression to activate the Wnt/β-catenin pathway, enhancing proliferation, migration, and epithelial-mesenchymal transition

doi: 10.1016/j.jbc.2025.108478

Figure Lengend Snippet: HKDC1 affects CRC cell cycle, apoptosis, and EMT via RCOR1 and activates the Wnt/β-catenin pathway .

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime Biotechnology) for 2 h and incubated overnight with primary antibodies: HKDC1 (1:1000, Abcam), RCOR1 (1:1000, Proteintech), Bax (1:2000, Proteintech), Bcl-2 (1:2500, Proteintech), CDK4 (1:1000, Proteintech), CDK6 (1:2000, Proteintech), Cyclin D1 (1:5000, Proteintech), β-catenin(1:5000, Proteintech), TCF4 (1:2000, Proteintech), c-Myc(1:2000, Proteintech), N-cadherin (1:5000, Proteintech), E-cadherin (1:50,000, Proteintech), and vimentin (1:1000, Proteintech).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: HKDC1 promotes colorectal cancer progression by regulating RCOR1 expression to activate the Wnt/β-catenin pathway, enhancing proliferation, migration, and epithelial-mesenchymal transition

doi: 10.1016/j.jbc.2025.108478

Figure Lengend Snippet: Immunofluorescence confirmed the interaction between HKDC1 and RCOR1, as well as the effect of HKDC1 regulation on the expression of EMT pathway proteins. A and B , the colocalization between HKDC1 and DLD-1 was visualized by a confocal laser scanning microscope in different cell lines. C and D , Immunofluorescence results demonstrate the impact of HKDC1 regulation and rescue experiment on the expression of EMT pathway-related proteins.

Article Snippet: Membranes were blocked with 5% non-fat milk (Beyotime Biotechnology) for 2 h and incubated overnight with primary antibodies: HKDC1 (1:1000, Abcam), RCOR1 (1:1000, Proteintech), Bax (1:2000, Proteintech), Bcl-2 (1:2500, Proteintech), CDK4 (1:1000, Proteintech), CDK6 (1:2000, Proteintech), Cyclin D1 (1:5000, Proteintech), β-catenin(1:5000, Proteintech), TCF4 (1:2000, Proteintech), c-Myc(1:2000, Proteintech), N-cadherin (1:5000, Proteintech), E-cadherin (1:50,000, Proteintech), and vimentin (1:1000, Proteintech).

Techniques: Immunofluorescence, Expressing, Laser-Scanning Microscopy

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