Journal: bioRxiv

Article Title: Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry

doi: 10.1101/2022.10.21.512986

Figure Lengend Snippet: (A) Immunohistochemistry (IHC) in serial CDX21 tissue sections for VM vessels (PAS + /CD31 - , pink), endothelial vessels (PAS + /CD31 + , pink+brown), REST, SYP and NCAM (brown). Scale bars, 50 µm. (B) Quantification of SYP, NCAM and REST expression in VM positive (black circles) and VM negative (open circles) regions of CDX identified in ( A ). Each circle represents a region that was defined and quantified, 2 independent mice were analyzed. n = 2 mice, n = 5 regions. Data are mean ± S.E.M. **p < 0.01, ****p < 0.0001 two-tailed unpaired student’s t-test. (C, D) Multiplex IHC showing VM vessels (PAS + /CD31 - , yellow arrows), endothelial vessels (PAS + /CD31 + , brown, green arrows) and REST (blue, in C ) or SYP (blue, in D ). Scale bars, 100 µm. (E) Percentage of VM vessels co-localized with REST (REST + VM vessels). n = 2 mice per CDX. Total REST expression for each CDX was derived from . Tumor sections from n = 3 independent mice per CDX were analyzed and the mean REST expression plotted (F) Pearson correlation of VM score versus REST transcript expression in CDX (R=0.57, p=0.0038). Average REST expression is shown for 3 independent tumors per CDX . (G) Pearson correlation of VM score versus SYP transcript expression in CDX (R=0.68, p=0.00027). Average SYP expression is shown for 3 independent tumors per CDX .

Article Snippet: RT–qPCR was performed using Taqman gene expression master mix and gene expression assays for ASCL1 (Hs00269932_m1), SYP (Hs00300531_m1), NCAM (Hs00941830_m1), CHGA (Hs00900370_m1), MYCL (Hs00420495_m1), HEY1 (Hs05047713_s1), REST (Hs05028212_s1), YAP1 (Hs00902712_g1), FOXC2 (Hs00270951_s1), MYC (Hs00153408_m1), ATOH1 (Hs00944192_s1), NEUROD1 (Hs01922995_s1) and ACTB (Hs01060665_g1) according to the manufacturer’s recommendations.

Techniques: Immunohistochemistry, Expressing, Two Tailed Test, Multiplex Assay, Derivative Assay

Journal: bioRxiv

Article Title: Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry

doi: 10.1101/2022.10.21.512986

Figure Lengend Snippet: (A) Representative immunoblots of CDX NE and non-NE cell lysates. n = 2-3 independent replicate tumors per CDX. Tubulin loading control and was run subsequently for each marker shown on the same blot (B) Representative brightfield images of tubule-forming assay with CDX NE and non-NE cells lysates. n = 2-3 independent replicate tumors per CDX. Scale bars, 500 µm. (C) Representative immunofluorescence of CDX non-NE cells in tubule-forming assay stained for human mitochondria (yellow) and nuclear DAPI (blue) in (B). n = 2-3 independent replicate tumors per CDX. Scale bars, 100 µm. (D-F) Representative images of Human umbilical vein endothelial cells (HUVECs) (D), CDX17 (E) and CDX30P (F) non-NE cells labelled with Cell Tracker Green forming hollow tubules when grown on Matrigel for 72 hours. Confocal microscopy images are shown after Z-stack software reconstruction (Imaris). Tubule length and diameter dimensions are shown, scale bars 50 µm. (G) Representative images of empty-vector control and NOTCH intracellular domain (NICD) expressing CDX31P cells three weeks post-doxycycline (dox) dox induction. Scale bars, 250 µm. (H) Percentage of adherent cells in control versus NICD expressing CDX31P cells three weeks post-dox induction in the suspension cells. n = 4 replicates from one tumor sample. Data are mean ± S.E.M. ***p < 0.001 two-tailed unpaired student’s t-test. (I) Representative immunoblots in control and NICD CDX31P cells ± dox. Tubulin loading control and was run subsequently for each marker shown on the same blot (J) Real-time quantitative PCR analysis of NE (ASCL1, SYP, NCAM, CHGA, MYCL) and non-NE (HEY1, REST, YAP1, FOXC2, MYC) markers in control versus NICD expressing CDX31P cells. Mean values are shown (black lines) where each circle represents one independent analysis, error bars are ± S.E.M (K) Representative images of tubule-forming assay with control and NICD CDX31P cells three weeks post-dox induction. Scale bars, 200 µm. n = 3 independent replicate tumors.

Article Snippet: RT–qPCR was performed using Taqman gene expression master mix and gene expression assays for ASCL1 (Hs00269932_m1), SYP (Hs00300531_m1), NCAM (Hs00941830_m1), CHGA (Hs00900370_m1), MYCL (Hs00420495_m1), HEY1 (Hs05047713_s1), REST (Hs05028212_s1), YAP1 (Hs00902712_g1), FOXC2 (Hs00270951_s1), MYC (Hs00153408_m1), ATOH1 (Hs00944192_s1), NEUROD1 (Hs01922995_s1) and ACTB (Hs01060665_g1) according to the manufacturer’s recommendations.

Techniques: Western Blot, Control, Marker, Immunofluorescence, Staining, Confocal Microscopy, Software, Plasmid Preparation, Expressing, Suspension, Two Tailed Test, Real-time Polymerase Chain Reaction

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: Journal of Neuroscience

Article Title: Casein Kinase 1 Suppresses Activation of REST in Insulted Hippocampal Neurons and Halts Ischemia-Induced Neuronal Death

doi: 10.1523/jneurosci.4045-13.2014

Figure Lengend Snippet: Figure1. CK1regulatesRESTbypromotingproteindegradation.a,Top,RepresentativeWesternblotshowingthattheCK1activatorpyrvinium,reducesRESTproteinabundanceinHeLacellsin a dose-dependent manner. Bottom, Summary data. b, Top, Representative Western blot showing that the CK1 inhibitor D4476 increases REST protein abundance in a dose-dependent manner. Bottom,Summarydata.c,RepresentativeWesternblotshowingthatpyrviniumreversestheincreaseinRESTelicitedbyD4476.Bottom,summarydata.d,Top,RepresentativeWesternblotshowing that overexpression of CK11, CK12, CK1, or CK1, but not CK11, CK12, or CK13, decreases REST protein abundance. Bottom, Summary data. e, Top, (Figure legend continues.)

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed with TaqMan probes (Applied Biosystems) for REST (Hs00958503_m1); GAPDH (Hs02758991_g1) served as an endogenous reference.

Techniques: Western Blot, Quantitative Proteomics, Over Expression

Journal: Journal of Neuroscience

Article Title: Casein Kinase 1 Suppresses Activation of REST in Insulted Hippocampal Neurons and Halts Ischemia-Induced Neuronal Death

doi: 10.1523/jneurosci.4045-13.2014

Figure Lengend Snippet: Figure 2. CK1 associates with and phosphorylates REST and increases -TrCP binding and REST ubiquitination. a, Diagram illustrating phosphorylated serine residues within two noncanonical degron motifs harbored within the REST C terminus that are critical to recognition by -TrCP. b, Coimmunoprecipitation of recombinant Myc from HeLa cells expressing Myc-CK1 and probed for REST showing that Myc-CK1 associates with endogenous REST. c, Immunoprecipitation of HA from N2A cells expressing WT or mutant HA-REST constructs incubated in the presence or absence of recombinant CK1 and - 32P-ATP. Top, Autoradiogram depicting phospho-HA-REST showing that single-point mutations S1013A, S1024A, and S1030A significantly alter CK1-dependent phosphorylation of REST. Bottom, Summary data. Values for phosphorylated HA-REST are normalized to values for HA-REST. d, Immunopre- cipitation of HA from N2A cells expressing WT HA-REST, double mutant HA-REST (E1009A/S1013A), and triple mutant HA-REST (S1024A/S1027A/S1030A) incubated in the presence or absence of recombinant CK1 and - 32P-ATP. Top, Autoradiogram depicting phospho-HA-REST shows that the double and triple HA-REST mutants abolish CK1-dependent phosphor- ylation. Bottom, Summary data. e, Immunoprecipitation of HA from N2A cells expressing WT HA-REST, double mutant HA-REST (S1013A/S1030A), or quadruple mutant HA-REST (S1013A/S1024A/S1027A/ S1030A) with an antibody to HA was incubated in the presence or absence of recombinant CK1 and - 32P-ATP. Top, Autoradiogram depicting phospho- HA-REST showing that the double and quadruple HA-REST mutants significantly decrease CK1-dependent phosphorylation of REST. Bottom, Summary data. f, Top, Immunoprecipitation of Flag from HeLa cells expressing myc-CK1 and Flag--TrCP subjected to Western blots probed for endogenous REST showing that CK1 increases the interaction between REST and -TrCP. Bottom, Immunoprecipitation of endogenous REST from HeLa cells expressing Myc-CK1 was subjected to Westerns and probed for REST, Myc-CK1, and ubiquitin showing that CK1 binds REST and promotes ubiquitination of REST. g, Top, Representative Western blot showing that two different sequences of shRNA-mediated knock down of -TrCP1/2 (-TrCP1/2shRNA-1,-TrCP1/2shRNA-2)inhippocampalneuronsincreaseRESTexpression.Bottom,Summarydata.n 3pertreatmentgroupin3independentexperiments.***p 0.001; **p 0.01; *p 0.05.

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed with TaqMan probes (Applied Biosystems) for REST (Hs00958503_m1); GAPDH (Hs02758991_g1) served as an endogenous reference.

Techniques: Binding Assay, Ubiquitin Proteomics, Recombinant, Expressing, Immunoprecipitation, Mutagenesis, Construct, Incubation, Phospho-proteomics, Western Blot, shRNA, Knockdown

Journal: Journal of Neuroscience

Article Title: Casein Kinase 1 Suppresses Activation of REST in Insulted Hippocampal Neurons and Halts Ischemia-Induced Neuronal Death

doi: 10.1523/jneurosci.4045-13.2014

Figure Lengend Snippet: Figure 3. REST is targeted to the proteasomal degradation pathway in neurons. a, Top, Representative Western blot showing that the proteasome inhibitor lactacystin injected in vivo increases REST abundance and global protein ubiquitination in the hippocampal CA1. Bottom, Summary data. b, Top, Representative Western blot showing that application of the proteasome inhibitor lactacystin applied to hippocampal neurons in vitro does not detectably alter -TrCP, but induces upregulation of REST anddownregulationoftheRESTtargetgeneGluA2undercontrolconditions.OGDdecreases-TrCP,increasesREST,anddecreases GluA2 in hippocampal neurons in vitro. Administration of lactacystin to neurons subjected to OGD does not further alter REST or GluA2. Bottom, Summary data. n 3 per treatment group in 3 independent experiments. c, Coimmunoprecipitation of endoge- nous REST from control hippocampal neurons or neurons subjected to OGD showing that endogenous REST associates with endogenous -TrCP under control conditions and that OGD promotes dissociation of REST from -TrCP. ***p 0.001.

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed with TaqMan probes (Applied Biosystems) for REST (Hs00958503_m1); GAPDH (Hs02758991_g1) served as an endogenous reference.

Techniques: Western Blot, Injection, In Vivo, Ubiquitin Proteomics, In Vitro, Control

Journal: Journal of Neuroscience

Article Title: Casein Kinase 1 Suppresses Activation of REST in Insulted Hippocampal Neurons and Halts Ischemia-Induced Neuronal Death

doi: 10.1523/jneurosci.4045-13.2014

Figure Lengend Snippet: Figure 5. Model depicting a hypothetical mechanism by which CK1 regulates REST abundance in differentiated neurons under physiological and pathological conditions. Under physiological conditions,CK1bindsandphosphorylatesRESTatsiteswithintwoneighboring,butdistinct,degronmotifs.Thephosphorylateddegrons(phospho-degrons)arecriticaltorecognitionofRESTbythe E3 ubiquitin protein ligase -TrCP, which ubiquitinates and targets REST for proteasomal degradation. Global ischemia reduces CK1 and -TrCP abundance, which leads to an increase in REST in thehippocampalCA1.RESTbindstotheRE1elementwithinthepromoteroftargetgenessuchasGluA2andorchestratestheassemblyofmSin3AandCoREST,HDACs1and2,G9a,andMeCP2.The REST–corepressorcomplexpromotesepigeneticremodelingofcorehistoneproteinsatthepromoteroftargetgenesandrepressestranscriptionofsynapticproteins.(Modifiedwithpermissionfrom Frescas and Pagano, 2008; Noh et al., 2012).

Article Snippet: Quantitative real-time PCR (qRT-PCR) was performed with TaqMan probes (Applied Biosystems) for REST (Hs00958503_m1); GAPDH (Hs02758991_g1) served as an endogenous reference.

Techniques: Ubiquitin Proteomics

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a – c REST induction in neurons that accumulate early tau pathology. a Immunolabeling of REST (green) the marker of early tau pathology, phospho-Ser202 tau (antibody CP13, red) and DNA (DAPI, blue) in the cortex (CTX) and the CA1 region of the hippocampus, shows elevated nuclear REST levels in neurons with accumulation of pSer202 tau in 11-month-old 3xTg mice b , c Quantification of nuclear REST levels in 11-month-old WT ( n = 4) and 3xTg ( n = 4) cortex ( b ) and 11-month-old WT ( n = 4) and 3xTg ( n = 6) hippocampus ( c ). d – f Loss of REST in aged 3xTg mice with advanced tau pathology. d Immunolabeling of REST (green), phospho-Ser396 tau, a marker of late, fibrillary, tau pathology (antibody PHF1, red) and DNA (DAPI; blue) in the cortex (CTX) and the CA1 region of the hippocampus, shows decreased nuclear REST levels in PHF1-positive neurons in 22-month-old 3xTg mice. Quantification of nuclear REST levels in the cortex ( e ) and hippocampus CA1 sector ( f ) of 22-month-old WT ( n = 3) and 3xTg ( n = 3) mice. g , h REST induction in J20 mice with early Aβ pathology. g Immunolabeling of REST (green), the neuron marker MAP2 (magenta) and nuclei (DAPI, blue) in the CA1 region of the hippocampus, shows elevated nuclear REST levels in 3-month-old J20 mice. h Quantification of nuclear REST levels in 3-month-old WT ( n = 7) and J20 ( n = 7) mice. i , j Loss of REST in aged J20 mice with advanced Aβ plaque pathology. i Immunolabeling of REST (green), Aβ (red) and DNA (DAPI, blue) in the CA1 region of the hippocampus, shows decreased nuclear REST levels in 18-month-old J20 mice. j Quantification of nuclear REST levels in 18-month-old WT ( n = 6) and J20 ( n = 6) mice. b , c , e , f , h , j Individual values (representing the average mean fluorescence intensities/mouse) as well as the mean ± S.E.M are shown. P -values were generated by one-way ANOVA with Tukey’s post-hoc test ( b , c , e , f ) or two-tailed unpaired t -test ( h , j ). a.u.-arbitrary units. Scale bars, 25 µm. Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: Immunolabeling, Marker, Fluorescence, Generated, Two Tailed Test

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a Immunolabeling of UPR activation (marker BiP/GRP78, red), REST (green), β-catenin (magenta) and DNA (DAPI, blue) in 11-month-old 3xTg and WT mice shows coordinate upregulation of BiP, nuclear REST and nuclear β-catenin expression in 3xTg mice. b Correlation between nuclear β-catenin and nuclear REST (left graph), BiP and nuclear REST (middle graph) and BiP and nuclear β-catenin (right graph) levels in the hippocampus CA1 region of 11-month-old 3xTg mice. Shown are the mean fluorescence intensity values for nuclear REST in individual CA1 neurons from n = 3 3xTg mice. a.u.- arbitrary units. The Pearson correlation coefficient (r) and P -value are shown. c Immunolabeling of REST (green) and neuron marker MAP2 (red) in 3xTg primary cortical neurons (PCNs) shows nuclear REST in neurons treated with vehicle and decreased nuclear REST in 3xTg neurons after a 24-h treatment with the Wnt/β-catenin inhibitors Dickkopf 1 (DKK1), XAV939 and ICG001, or the PERK inhibitor GSK2606414. d Quantification of average nuclear REST levels in WT PCNs, as well as 3xTg PCNs treated with vehicle or the individual drugs. The P -values for planned comparisons (using two-tailed unpaired t -test) between each treated group and 3xTg treated with vehicle are shown; for WT vs 3xTg/vehicle, P < 10 −4 . e Immunolabeling of REST (green) and neuron marker MAP2 (red) in WT primary cortical neurons (PCNs) shows low nuclear REST in WT neurons treated with vehicle or the UPR inducer thapsigargin (TG), mildly increased nuclear REST expression after treatment with the GSK3β inhibitors CHIR99021 (Chiron) and lithium chloride (Li), and strongly increased nuclear REST levels after treatment with a combination of TG and Chiron, or a combination of TG and Li. f Quantification of average nuclear REST levels. P -values for pre-planned comparisons, using two-tailed unpaired t -tests, are indicated. d , f Nuclear REST levels are expressed as mean fluorescence intensity/nucleus (in arbitrary units, a.u.) for n = 5 (all drugs except DKK-1) or n = 3 ( f , and DKK-1 in panel e ) independent experiments. Individual values and the mean ± S.E.M are shown. Scale bars, 25 µm. Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: Immunolabeling, Activation Assay, Marker, Expressing, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a , b Loss of REST in excitatory neurons increases CDK5 expression in cortex and hippocampus. a Immunolabeling for CDK5 (green) and the neuronal marker MAP2 (magenta) in CA1 neurons of the hippocampus in 9-month-old 3xTg and 3xTg;cKO mice. b Quantification of CDK5 immunofluorescence intensity in the hippocampus and cortex of 9-month-old 3xTg ( n = 4) and 3xTg;cKO ( n = 4) mice. c , d Loss of a single REST allele increases CDK5 expression. c Immunolabeling for CDK5 (green) and MAP2 (magenta) in 29-month-old 3xTg and 3xTg;GT (heterozygous REST null) mice. d Quantification of CDK5 immunofluorescence intensity in 28–29-month-old 3xTg ( n = 6) and 3xTg;GT ( n = 6) mice. e , f Loss of REST in excitatory neurons increases GSKβ expression in cortex and hippocampus. e Immunolabeling for GSK3β (green) and MAP2 (magenta) in hippocampal CA1 neurons in 9-month-old 3xTg and 3xTg;cKO mice. f Quantification of GSK3β immunofluorescence intensity in 9-month-old 3xTg ( n = 4) and 3xTg;cKO ( n = 4) mice. g , h Loss of a single REST allele increases GSK3β expression. g Immunolabeling for GSK3β (green) and MAP2 (magenta) in 29-month-old 3xTg and 3xTg;GT (heterozygous REST null) mice. h Quantification of GSK3β immunofluorescence intensity) in 28–29-month-old 3xTg ( n = 6) and 3xTg;GT( n = 6) mice. i Western blot analysis of CDK5 and GSK3β levels in the hippocampus of 29-month-old 3xTg and 3xTg;GT mice. j Quantification shows CDK5 and GSK3β levels normalized to actin in 28–29-month-old 3xTg ( n = 5) and 3xTg;GT ( n = 5) mice. b , d , f , h , j Individual values and the mean ± S.E.M are shown; P -values are derived from two-tailed unpaired t -tests. k , m Immunofluorescence labeling of REST (green) and CDK5 (red, k ) or GSK3β (red, m ) in a NCI case with early AD pathology shows an inverse relationship between nuclear REST and either CDK5 ( k ) or GSK3β ( m ) total cellular levels in neurons of the prefrontal cortex. l , n Correlation between nuclear REST levels and total cellular levels of CDK5 ( l ) or GSK3β ( n ) in individual neurons from n = 3 NCI cases with early pathology. Shown are the Pearson r correlation coefficients and the P -values. Scale bars, 25 μm. Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: Expressing, Immunolabeling, Marker, Immunofluorescence, Western Blot, Derivative Assay, Two Tailed Test, Labeling

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a Cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses leading to either REST inhibition (short hairpin RNA; sh4) or overexpression (human REST cDNA: hREST ), as previously described . Expression of γ-secretase components was assessed by qRT-PCR. b REST lx/lx MEF cells were transduced with Cre recombinase (generating REST-KO lines 1–3) or a control vector without Cre (lines WT 1–3). The REST floxed ( REST lx ) and Cre-recombined REST ( REST rec ) alleles were detected by PCR. No REST lx band was detected in REST-KO cells, suggesting complete Cre -mediated recombination. c qRT-PCR using two sets of primers spanning the transcript shows loss of REST mRNA in REST-KO MEFs. d Immunolabeling with anti-REST antibody (REST14 ; white) shows loss of REST expression in REST-KO MEFs. Nuclei are labeled with DAPI (blue). Scale bar, 40 μm. e Western blot analysis of γ-secretase components in WT and REST-KO MEFs. The transferrin receptor (TfR) served as loading control. f Quantification of protein levels normalized to TfR shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). For PS1, similar results were seen with antibodies against the N-terminus (NTF; antibody 231f) or C-terminus (CTF; antibody EP2000Y). g REST-KO MEFs show elevated γ-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99-FLAG in the presence or absence of a γ-secretase inhibitor (+I), and levels of AICD-FLAG were determined by Western blotting using TfR as a loading control. h Quantification of γ-secretase activity in membrane preparations from WT and REST-KO cells. AICD-FLAG levels were normalized to TfR, and shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). Loss of REST in REST-KO MEFs leads to elevated Aβ40 ( i ) and Aβ42 ( j ) levels following transfection of hAPP WT or hAPP Swe . Lentiviral transduction of human REST cDNA (hREST) suppresses Aβ production. Individual values and the mean ± S.E.M are shown for n = 6 ( a ) or n = 3 ( c , f , h , i , j ) independent experiments. P -values are derived from two-tailed unpaired t -tests ( a , c , f , h ) or one-way ANOVA with Tukey’s post-hoc test ( i , j ). Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: Cell Culture, Transduction, Recombinant, Inhibition, shRNA, Over Expression, Expressing, Quantitative RT-PCR, Control, Plasmid Preparation, Immunolabeling, Labeling, Western Blot, Activity Assay, Incubation, Membrane, Transfection, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a , b Increased mTau and pTau accumulation in REST-deficient 3xTg mice. a Immunolabeling for conformationally altered tau (antibody MC1, mTau) or pSer202 tau (antibody CP13, right panels, pTau), and DNA (DAPI) in 18-month-old 3xTg and 3xTg;cKO mice with a CA1 neuron-specific REST deletion. b Quantification of mTau-positive and pTau-positive neuron density in the hippocampal CA1 sector of 17–18-month-old 3xTg ( n = 15), 3xTg;cHET ( n = 8) and 3xTg;cKO ( n = 3) mice. Scale bar, 25 μm. c Quantification of pTau-positive neuron density in 17–18-month-old 3xTg ( n = 8), 3xTg;cHET ( n = 8) and 3xTg;cKO ( n = 10) mice generated with a second Cre line that gives rise to conditional forebrain deletion of REST in glutamatergic neurons. d , e REST deletion induces multiple phospho-tau epitopes associated with neurofibrillary pathology. d Western blot analysis of pThr231-tau (antibody AT180) and pSer396-tau (antibody PHF1), as well as total tau (antibody tau-5) and actin, in the hippocampus of 9-month-old 3xTg ( n = 5) and 3xTg;cKO ( n = 4) mice. e Quantification of pThr231-tau:total tau, pSer396-tau:total tau and total tau:Actin ratios in 9-month-old 3xTg and 3xTg;cKO mice. f , g Increased Aβ plaque deposition in aged REST-deficient J20 mice. f Immunolabeling of Aβ (white) in 14-month-old J20 and J20;cKO hippocampus and cortex. g Quantification of Aβ plaque burden in the hippocampus (left bar graph) and the cortex (right bar graph) in 12−14-month-old J20 ( n = 26), J20;cHET ( n = 8), and J20;cKO ( n = 15) mice. Scale bar, 200 μm. b , c , e , g Individual values as well as the mean ± S.E.M are shown. P -values were generated by one-way ANOVA with Tukey’s post-hoc test ( b , c ), Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test ( g ) or two-tailed unpaired t -test ( e ). Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: Immunolabeling, Generated, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a , b Increased neurodegeneration in REST-deficient 3xTg mice. a TUNEL labeling (green), immunolabeling for p-Ser202 tau (pTau) (antibody CP13, red), and DAPI labeling for DNA identifies TUNEL-positive nuclei in neurons with NFT-like tau pathology in hippocampal CA1 (top 2 panels) and CA3 (lower 2 panels) sectors in 29-month-old 3xTg;GT but not 3xTg mice. b Quantification of TUNEL-positive cells in 27–29-month-old WT ( n = 4), GT ( n = 3), 3xTg ( n = 6) and 3xTg;GT ( n = 6) mice. P-values were generated by two-way ANOVA with Tukey’s post-hoc test. Scale bar, 25 μm. c Time course of spatial learning in the Morris water maze for 17–18-month-old mice of the indicated genotypes. d Memory retrieval in the probe trial of the Morris water maze. Shown are the numbers of entries in the target (platform) area and time spent in target (platform) area for 17–18-month-old mice of the indicated genotypes. Two-way ANOVA revealed no significant interaction between REST genotype (+/+, cHET) and AD pathology (WT, 3xTg): F (1, 51) = 2.41, P = 0.12. One-way ANOVA with Tukey’s post-hoc test was then conducted and the P-values are shown. Panels c , d : n = 17 WT, n = 11 REST cHET, n = 16 3xTg, and n = 11 3xTg;cHET mice. e Time course of spatial learning in the Morris water maze for 12–14-month-old mice of the indicated genotypes. REST loss-of-function alleles do not further impair learning in J-20 mice. f Memory retrieval in the probe trial for 12–14-month-old mice of the indicated genotypes. J-20 mice have impaired memory retrieval relative to WT mice by two-tailed Mann–Whitney U test. Two-way ANOVA revealed no significant interaction between REST genotype (+/+, cHET, cKO) and AD pathology (WT, J20): F (2,100) = 0.82, P = 0.44. Kruskal–Wallis one-way ANOVA with Dunn’s post-hoc test was then conducted and the P -values are shown. Panels e , f: n = 17 WT, n = 14 REST cHET, n = 18 REST cKO, n = 31 J20, n = 8 J20;cHET and n = 18 J20;cKO mice. b , d , f Shown are individual values and the mean ± S.E.M. c , e Shown are the mean ± S.E.M, and P -values for planned comparisons by the two-tailed unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: The qualitative and quantitative pattern of REST immunolabeling for the REST Bethyl IHC-00141 antibody was very similar to those obtained with 2 independent REST antibodies by immunofluorescence .

Techniques: TUNEL Assay, Labeling, Immunolabeling, Generated, Two Tailed Test, MANN-WHITNEY