Journal: Nature neuroscience

Article Title: NCOR1/2 loss of function impairs memory through a novel GABAergic hypothalamus–CA3 projection

doi: 10.1038/s41593-018-0311-1

Figure Lengend Snippet: . (a) Patients carrying copy number variants (CNV) or single nucleotide variants (SNV) in NCOR1 , NCOR2 , or HDAC3 . Genomic coordinates are shown in hg19. DDD_SNV, single nucleotide variants retrieved from Deciphering Developmental Disorders (DDD) website (United Kingdom); kb, kilobase. (b-d) Schematic representations for the deletions and point mutations affecting NCOR1 , NCOR2 , or HDAC3 , respectively, observed in patients with neurodevelopmental disorders. The locations of deletions are depicted in red, and the point mutations in pink. (e) Western blot of HEK-293 cells transfected with plasmids expressing wild-type (WT) HDAC3 with or without mutant L266S. The experiment was repeated independently once with similar results. The blot images have been cropped. (f) Fluorescence-based HDAC enzyme assay after anti-HDAC3 immunoprecipitates from cell lysates overexpressing the indicated HDAC3 proteins. Box plots center line, median; box limits, upper and lower quartiles; whiskers, minimal and maximum values. Data were analyzed by two-tailed unpaired t test. n=4 biological independent samples for each group. (g) Western blot of HEK-293 cells transfected with plasmids expressing WT HDAC3, WT NCOR1, with or without the NCOR1 deletion mutant (Del). The experiment was repeated independently once with similar results. Data were analyzed by two-tailed unpaired t test. n=3 biological independent samples for each group. The blot images have been cropped. (h) Chromatin immunoprecipitation (ChIP) with anti-HDAC3 antibodies followed by qPCR using primers targeting promoters of the indicated genes ARNTL and CDKN1A . RPLP0 serves as a negative control. Data is expressed as mean ± S.E.M. For detailed statistics results, see . * P ≤ 0.05 is set as significance.

Article Snippet: For western blot, immunoprecipitates and total tissue lysates were resolved by Tris-glycine SDS-PAGE, transferred to PVDF membranes, and blotted with antibodies against NCOR1 (lab-made) , TBLR1 (IMGENEX, IMG591), and HDAC3 (Abcam 7030).

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Fluorescence, Enzymatic Assay, Two Tailed Test, Chromatin Immunoprecipitation, Negative Control

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) CMA activity in 5 human NSCLC cell lines and lung epithelial BEAS-2B cells. CMA activity was measured using the KFERQ-PS-Dendra reporter assay (left) and quantified as fluorescent puncta per cell (right). Representative images (center) of cells expressing KFERQ-PS-Dendra in red and nuclei highlighted with DAPI. Inserts show higher magnification of the red channel. n ≥ 105 cells in three independent experiments. (**** P ≤ 0.0001). Created in BioRender. https://BioRender.com/a8dxxz3 . ( B ) Heatmap of the transcriptional differences of CMA-related genes (left) and calculated CMA score (right) in the same cell lines as ( A ). n = 3 independent experiments. (** P = 0.0013, ** P = 0.0051, * P = 0.0283, ** P = 0.0019, *** P = 0.0001). ( C ) Immunofluorescence staining for NCoR1 and RARα in the indicated cell lines. Top: representative images of single or merged (overlay) channels with nuclei highlighted with DAPI. Full field images are shown in Appendix Fig. ). Bottom: quantification of nuclear intensity for each protein. n ≥ 158 cells in three independent experiments (*** P = 0.0004, **** P ≤ 0.0001). ( D ) CMA activity in A549 cells expressing KFERQ-PS-Dendra upon transfection with control (Ctrl) siRNA or siRNA targeting RARα. Representative images (left) as in ( A ) and quantification of fluorescent puncta per cell (right). n ≥ 212 cells from six independent experiments (* P = 0.0317). ( E ) CMA activity in A549 cells expressing KFERQ-PS-Dendra and maintained in serum-free media supplemented with 10 µM ATRA or equal volume vehicle (DMSO). n ≥ 87 cells from three independent experiments. Representative images are shown in Fig. . (**** P ≤ 0.0001). ( F ) CMA activity in A549 cells expressing KFERQ-PS-Dendra and maintained in media supplemented with 5 µM AM580 or equal volume vehicle (DMSO). n ≥ 72 cells from two independent experiments. Representative images are shown in Fig. . (*** P = 0.0007). ( G ) CMA activity in A549 cells expressing KFERQ-PS-Dendra upon transfection with control (Ctrl) siRNA or siRNA targeting NCoR1. Representative images (left) as in ( A ) and quantification of fluorescent puncta per cell (right). n ≥ 124 cells from four independent experiments. (**** P ≤ 0.0001). ( H ) CMA activity in A549 cells expressing the KFERQ-PS-Dendra upon overexpression of either wild-type (WT) RARα or the AHT RARα mutant. Representative images (left) as in ( A ) and quantification of number of fluorescent puncta per cell (right) n ≥ 45 cells from three independent replicates (* P = 0.0492). Data information: All values are mean + SEM, with individual data points when n < 10. Ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test ( A–C ) or unpaired two-tailed t test ( D–H ) were used. .

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Activity Assay, Reporter Assay, Expressing, Immunofluorescence, Staining, Transfection, Control, Over Expression, Mutagenesis, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) Table of our five NSCLC cells lines, their CMA activity, and status of oncogenes. ( B ) Representative immunoblot for LAMP2A (left) and quantification of LAMP2A levels (right) in the indicated NSCLC cell lines and non-tumorigenic control BEAS-2B cells normalized to levels of BEAS-2B cells in each experiment (right). n = 5–6 independent experiments. (**** P ≤ 0.0001). ( C–F ) Transcriptional analysis of CMA-related genes ( C ), calculated CMA z-score ( D ), expression (as z-score) of CMA effectors, positive regulators, and negative regulators ( E ), and NCoR1/RARα ratio ( F ), in 17 non-tumorigenic lung cell lines and 15 NSCLC-derived cell lines. The heat map in ( C ) shows differential gene expression. Cell lines are listed in Appendix Table . Data from (Hruz et al, ). ( D ): ** P = 0.0031; ( E ): *** P = 0.0001, **** P ≤ 0.0001, * P = 0.0403, *** P = 0.0004, *** P = 0.0005, * P = 0.0108, ** P = 0.0012; ( F ): ** P = 0.0029). Data information: All values are mean + SEM or individual data points to represent individual samples. One-way ANOVA ( B ), unpaired two-tailed t test ( D , F ), or multiple unpaired t tests with individual variances and Bonferroni post-hoc analysis ( E ) were performed. are available online for this figure.

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Activity Assay, Western Blot, Control, Expressing, Derivative Assay, Gene Expression, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) RARα protein levels and RNA expression in A549 cells transfected with control (ctrl) or RARα siRNA. Representative immunoblot (left), protein level quantification (center), and RNA expression normalized to control siRNA (right) are shown. n = 4 independent experiments. (* P = 0.0107, ** P = 0.0081). ( B ) Representative fluorescence images of A549 cells expressing KFERQ-PS-Dendra treated with 10 µM ATRA or equal volume DMSO in serum-free media for 24 h. Quantification is shown in Fig. . ( C ) Representative fluorescence images of A549 cells expressing KFERQ-PS-Dendra treated with 5 µM AM580 or equal volume DMSO for 24 h. Quantification is shown in Fig. . ( D ) NCoR1 protein levels and RNA expression in A549 cells transfected with control (ctrl) or NCoR1 siRNA. Representative immunoblot (left), protein level quantification (center), and RNA expression normalized to control siRNA (right) are shown. n = 3 independent experiments. (** P = 0.0028, * P = 0.0400). ( E ) Representative immunoblot (left) of A549 cells expressing KFERQ-PS-Dendra transfected with equal amounts of DNA of WT RARα or AHT RARα. ( F ) Schematic of mCherry-GFP-LC3 reporter (Kimura et al, ). When the reporter is associated to autophagosomes, both mCherry and GFP fluoresce and autophagosomes are visualized as yellow puncta. Once the autophagosome fuses with a lysosome GFP fluorescence is quenched and autophagolysosomes appear as red fluorescent puncta. Created in BioRender. https://BioRender.com/oqvzi51 . ( G , H ) Macroautophagy activity in A549 cells expressing mCherry-GFP-LC3 treated with 10 µM ATRA ( G ) or 5 µM AM580 ( H ) or equal volume DMSO (No treatment; NT) for 24 h. Representative images (left) and quantification of number of the indicated autophagic compartments (right). AV: autophagic vacuoles (total number of fluorescent red puncta), APG: autophagosomes (total number of green fluorescent puncta) and AUT: autolysosomes (total number of red – green fluorescent puncta). n = 35–45 cells from 2 independent experiments. ( G : ** P = 0.0086, ns = not significant, ** P = 0.0098, H : **** P ≤ 0.0001, * P = 0.0448, ** P = 0.0015). Data Information: All values are mean + SEM. Unpaired two-tailed t test ( A , D ) and two-way ANOVA ( G , H ) were used. Insets show higher magnification and nuclei are highlighted with DAPI. are available online for this figure.

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: RNA Expression, Transfection, Control, Western Blot, Fluorescence, Expressing, Activity Assay, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) Flowchart of the computational screen to identify small molecules predicted to bind the inactive (NCoR1-bound, PDB ID: 3KMZ) or active (SRC-bound, PDB ID: 3KMR) conformation of RARα. Structure of NCoR1-bound RARα used in the screen and docked compounds bound into NCoR1 site are shown on the left. ( B ) CMA activity in mouse fibroblasts (NIH-3T3 cells) expressing KFERQ-PS-Dendra, 24 h after treatment with 10 µM of compounds identified from ( A ), with each data point representing an individual compound. n = 1500–4000 cells imaged across nine fields. CMA activity is shown as z-score of the fluorescent puncta per cell values with potential inhibitors identified by z-score < −1 (blue dots below dashed line). ( C , D ) CMA activity in A549 cells expressing KFERQ-PS-Dendra treated with 1 µM of each putative CMA inhibitor compound or DMSO for 24 h. Representative images ( C ) and quantification of fluorescent puncta per cell ( D ). n ≥ 40 cells. (**** P ≤ 0.0001, * P = 0.0302). ( E ) CMA activity in A549 cells expressing KFERQ-PS-Dendra treated with increasing concentrations of CIM7. Representative images (top) and quantification of fluorescent puncta per cell (bottom). Dashed line indicates CMA activity in untreated cells. n > 96 individual cells from three to four independent experiments. (**** P ≤ 0.0001, *** P = 0.0009). ( F ) CMA activity in non-cancer cells treated with 5 µM CIM7 or equal volume DMSO. Representative image of BEAS-2B cells (top; Images for other cell lines are in Appendix Fig. ) and quantification of fluorescence puncta per cell (bottom). n > 20 individual cells. (ns = not significant). ( G ) Representative immunoblot for Dendra in A549 cells treated with the indicated concentrations of CIM7 or DMSO in the absence or presence of ammonia chloride and leupeptin (N/L) (top) and densitometric quantification (below). Ponceau staining is shown as loading control. n = 4 independent experiments. ( H ) Heatmap of the z-scores of the transcriptional differences of CMA-related genes (left) and calculated CMA score (right) in A549 cells at the indicated times after addition of 5 µM CIM7. n = 3 independent experiments. (** P = 0.0061). ( I ) Time course of changes in CMA activity calculated by number of fluorescent puncta per cell in A549 cells treated with 5 µM CIM7. n > 95 cells from three independent experiments. (*** P = 0.0008, **** P ≤ 0.0001). Data information: All values are mean + SEM, with individual data points in bar graphs when n < 10. Ordinary one-way ANOVA followed by Bonferroni’s multiple comparisons post-hoc test ( D , E , H , I ), or non-linear regression ( E , I ), or multiple t test ( F ) were used. .

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Activity Assay, Expressing, Fluorescence, Western Blot, Staining, Control

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) Molecular dynamic simulations of CIM7 (white sticks) bound to the inactive RARα (cyan and blue, with sidechains of interacting residues in green) in the absence of NCoR1 peptide reveals a shift from the inactive conformation (left, 0 ns) to the active RARα conformation (right, 1000 ns) based on helix H11 (blue). ( B ) Representative isothermal titration calorimetry curve of CIM7 with recombinant RARα LBD showing enthalpy (ΔH) with increasing molar ratio. This experiment was repeated five times with consistent results. ( C ) Molecular dynamic simulations of CIM7 (white) bound to the inactive RARα (cyan) in the presence of NCoR1 peptide (magenta) (top, 0 ns) reveals a disruption of the b-sheet/b-sheet interaction necessary for NCoR1 to bind RARα conformation over time (bottom, 1000 ns). ( D ) The predicted distance from T2050 Cα atom on NCoR1 (red in C ) and R394 Cα atom on RARα (blue in C ) in simulations with CIM7 present or absent. n = 3–4 individual simulations. (**** P ≤ 0.0001). ( E ) The EC 50 (µΜ) of NCoR1 binding to RARα, determined by fluorescence polarization anisotropy (FPA) with RARα and the NCoR1 peptide in the presence of 25 µΜ of CIM7 or DMSO. n = 2–3 technical replicates. (**** P ≤ 0.0001). ( F ) TR-FRET evaluation of SRC binding to recombinant RARα with increasing concentrations of CIM7. n = 4 technical replicates. ( G ) FPA with RARα and the NCoR1 peptide in the presence of 10 µM ATRA or DMSO. n = 2–3 technical replicates. ( H ) TR-FRET evaluation of SRC binding to recombinant RARα with increasing concentrations of ATRA. n = 4 technical replicates. ( I ) Schematic representation of the differences between CIM7 and ATRA effect on coregulator binding to RARα. Created in BioRender. https://BioRender.com/qbb0uvv . Data information: All values are mean + SEM. Ordinary two-way ANOVA ( D , E ) and non-linear regression ( E–H ) were used. .

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Isothermal Titration Calorimetry, Recombinant, Disruption, Binding Assay, Fluorescence

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) CMA activity in A549 cells expressing KFERQ-PS-Dendra with control (Ctrl) or targeted siRNA for RARα or NCoR1 after treatment with 5 µM of CIM7 for 24 h relative to untreated cells. Representative images (left) with KFERQ-PS-Dendra in red and nuclei highlighted with DAPI (inserts show higher magnification of the red channel) and quantification of fluorescent puncta per cell normalized to untreated cells in each siRNA background (right). n ≥ 115 cells in three independent experiments. (** P = 0.0023, **** P ≤ 0.0001). ( B ) Proximity ligation assay (PLA) of NCoR1 and RARα in A549 cells following 4-h treatment with DMSO or 5 µM CIM7. Representative images (left) and quantification of mean fluorescent intensity (MFI) of the NCoR1/RARα complex as visualized in red. Nuclei are highlighted with DAPI. n ≥ 95 cells in two independent experiments. (**** P ≤ 0.0001). ( C ) Representative immunoblot for RARα of streptavidin pulldowns (left) and flowthrough (right) of A549 cellular lysate incubated without additions or with biotin-CIM7 (50 µM) and/or CIM7 (250 µM or 500 µM). n = 3 independent experiments. ( D ) Representative immunoblot for RARα of streptavidin pulldowns (left), flowthrough (center), and input (right) of cellular lysate from A549 cells expressing AHT or wild-type (WT) RARα incubated without additions or with biotin-CIM7 (50 µM). n = 3 independent experiments. ( E ) Heatmap of z-score for expression of genes changing significantly upon 3 h treatment with CIM7 in A549 cells expressing WT RARα and cells expressing AHT RARα. ( F ) Gene Ontology enrichment of all genes based on expression fold change of CIM7 treatment compared to DMSO, as predicted by STRING analysis. Bar coloring corresponds to false discovery rate (FDR). ( G ) TRANSFAC and JASPAR PWMs obtained through analysis with Enrichr sorted by rank-based ranking of genes significantly altered ( >1.5-fold or <0.5-fold change versus DMSO, P < 0.05 based on unpaired two-tailed t test) upon CIM7 treatment in the presence of WT RARα in A549 cells. Data information: All values are mean + SEM. Unpaired two-tailed t test ( A , B ) was used. .

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Activity Assay, Expressing, Control, Proximity Ligation Assay, Western Blot, Incubation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A ) Representative images (left) and quantification (right) of immunohistochemistry (IHC) for NCoR1 in a panel of 10 healthy lung and 39 NSCLC tumor samples. (**** P ≤ 0.0001). ( B ) Representative images (left) and quantification (right) from the same tissues as in ( A ) of IHC for RARα. ( P = 0.2784). ( C ) Ratio of NCoR1 to RARα nuclear intensities from the IHC of tissues in ( A , B ), n = 9 healthy lung and 39 NSCLC tumor samples total. (*** P = 0.0002). ( D ) CMA score (top) and NCoR1/RARα ratio (bottom) across 30 cancer types based on data from the TCGA. CMA score is calculated as a comparison between the cancers. ( E ) Correlation between NCoR1/RARα ratio and CMA score in the 30 cancer types from ( D ). ( F ) CMA score (left) from ( D ) and NCoR1/RARα ratio (right) from ( E ) in patient tumors with wild-type (WT) or mutant (Mut) TP53 based on data from the TCGA. n > 1500 individual tumors. (**** P ≤ 0.0001). Data information: All bar values are mean + SEM and dots represent individual samples, except in ( E ) where dots represent average expression in each cancer type. Unpaired two-tailed t test ( A–C , F ) and simple linear regression ( E ) were used. .

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: Immunohistochemistry, Comparison, Mutagenesis, Expressing, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: Small molecule disruption of RARα/NCoR1 interaction inhibits chaperone-mediated autophagy in cancer

doi: 10.1038/s44321-025-00254-y

Figure Lengend Snippet: ( A , B ) CMA score ( A ) and NCoR1/RARα ratio ( B ) calculated from RNA-seq of tumor and surrounding healthy lung tissue from NSCLC patients. Data from (Sanchez-Palencia et al, ). n > 44 individual patients. ( A ): * P = 0.0248, ( B ): ** P = 0.0029). ( C ) Correlation of NCoR1/RARα ratio and CMA score in select cancers. Each individual data point represents an individual patient sample. Data from the TCGA Pan Cancer Altas (Liu et al, ). n > 8 individual tumors. ( D–G ) CMA score (left) and NCoR1/RARα ratio (right) calculated in tumors with wild-type (WT) or mutant (Mut) KRAS ( D ), MYC ( E ), BRCA1 ( F ), or BRCA2 ( G ). Data from the TCGA Pan Cancer Altas (Liu et al, ). n > 38 individual tumors. (**** P < 0.0001, ns = not significant). Data information: All bar values are mean + SEM. Unpaired two-tailed t test ( A , B , D – G ) and simple linear regression ( C ) were used. are available online for this figure.

Article Snippet: NCoR1 siRNA , Origene , Cat# SR423484A.

Techniques: RNA Sequencing, Mutagenesis, Two Tailed Test

Journal: Frontiers in Behavioral Neuroscience

Article Title: Neural Androgen Receptors Modulate Gene Expression and Social Recognition But Not Social Investigation

doi: 10.3389/fnbeh.2016.00041

Figure Lengend Snippet: Names and assay numbers of the investigated genes.

Article Snippet: Ncor1 , Nuclear receptor co-repressor 1 , Mm00448681_m1.

Techniques: Binding Assay