Fig 1: LSD1 is enriched at the cell surface and in intracellular compartments of ACE2-expressing cells.a The receptor-binding domain (RBD) sequence of SARS-CoV-2 showing the critical residue (Q; glutamine 493) that binds to ACE2 lysine 31 and the conservation of this sequence in different species. In silico prediction35 gave a probability of 0.7 of a methylation/demethylation signature at lysine 31. b FACS tSNE analysis of cell surface and intracellular expression of ACE2 and LSD1 in Caco-2 cells. The bar chart indicates the percentage ACE2+ or LSD1+ cells in the total Caco-2 population, also shown in the FACS dot plots (ACE2+ cells in blue and LSD1+ cells in red). Data are mean ± SEM (n = 3). c Western blot analysis of ACE2 and LSD1 cell surface expression by Caco-2 cells. Membrane lysates were analyzed by SDS-PAGE and blotted for ACE2, LSD1, and integrin-ß1. d Western blot analysis of LSD1 IP samples. Following LSD1 IP of Caco-2 lysates, samples were analyzed by SDS-PAGE and blotted for CoREST (66kD) and HDAC2 (60kD). e Dot plot quantification of the fluorescence intensity (cell surface and cytoplasmic) of ACE2 and LSD1 in Caco-2 and MRC-5 cells. >50 cells were analyzed for each group. The Pearson correlation coefficient (PCC) was calculated for LSD1 and ACE2 colocalization (n = 20 cells analyzed). Mann-Whitney test: *P < 0.05, ****P < 0.0001 denote significant differences. f Duolink® proximity ligation assay measurements of protein interactions were performed on unpermeabilized Caco-2 cells transfected with either VO construct or an LSD1 WT plasmid followed by treatment with 0, 1, or 10 ng of SARS-CoV-2 spike protein. The Duolink assay produces a single bright dot per interaction within the cell. Representative images (top) are shown for ACE2 and SARS-CoV-2 Spike Duolink®. PLA signal intensity of the Duolink® assay (bottom) is shown for average dot intensity (single Duolink dot). Data represents n = 20 cells, with significant differences calculated using the Kruskal-Wallis ANOVA (*P < 0.05, **P < 0.01, ****P < 0.0001). Representative images are shown with 10 µM scale bar in orange. g Schematic of SARS-CoV-2 infection assays. Caco-2 cells were seeded 24 h before the experiment. Then, cells were infected with SARS-CoV-2 (MOI 1.0). After 1 h viral adsorption incubation, the virus inoculum was removed and drug-free medium was added. Then, cell culture supernatants were harvested at 0, 24, 48, or 72 hpi (hour post-infection) and infected cells collected at 24, 48, or 72 hpi. Viral genomes were detected in the extracted RNA by qRT-PCR, and viral protein was quantified by digital pathology (ASI system). h qRT-PCR analysis to detect the growth kinetics of SARS-CoV-2 in Caco-2 and MRC-5 culture supernatants at the indicated time points after viral infection. The quantity of viral genomes is expressed as log10 TCID50 equivalents/mL. Data are mean ± SEM (n = 3). One-way ANOVA; *P < 0.05, **P < 0.01 denote significant differences. i FACS analysis of expression of SARS-CoV-2 N (nucleocapsid), ACE2, and LSD1 in uninfected vs. SARS-CoV-2-infected Caco-2 cells (n = 3, unpaired t-test: ***P < 0.001). j Dot plot quantification of the fluorescence intensity (cell surface and cytoplasmic) of ACE2 and LSD1 in uninfected or SARS-CoV-2-infected CaCo-2 and MRC-5 cells. >50 cells were analyzed for each group. Mann–Whitney test: n.s. denotes non-significant, ***P < 0.001, ****P < 0.0001. The PCC was calculated to assess colocalization in MRC-5 or Caco-2 cells with/without infection (n = 20 cells analyzed). Data are mean ± SEM. Mann-Whitney test: n.s. denotes non-significant, ****P < 0.0001 denote significant differences. k Representative image of uninfected or SARS-CoV-2-infected Caco-2 cells using the Andor WD Revolution Inverted Spinning Disk microscopy system. Cells were not permeabilized (surface), with immunostaining for ACE2, LSD1, and SARS-CoV-2 N (nucleocapsid). DAPI (blue) was used to visualize nuclei. Scale bar, 12 µm (inset). l Dot plot quantification of the fluorescence intensity of H3K9me2 and H3K4me2 in uninfected or SARS-CoV-2-infected Caco-2 cells. >50 cells were analyzed for each group. Mann–Whitney test: ****P < 0.0001 denote significant differences. m Dot plot quantification of the fluorescence intensity (cell surface and intracellular) of SETDB1, G9A, and ACE2 in uninfected or SARS-CoV-2-infected Caco-2 cells. >50 cells were analyzed for each group. Mann-Whitney-test: n.s. denotes non-significant, ***P < 0.001, ****P < 0.0001 denote significant differences. n Representative image of uninfected or SARS-CoV-2 infected human biliary epithelial cells (HBECs) using the ASI digital pathology system and immunostaining for a-tubulin, Muc5ac, KRT5, LSD1, ACE2, and SARS-CoV-2 N (nucleocapsid). DAPI (blue) was used to visualize nuclei. Scale bar, 12 µm (inset). Dot plot quantification of the mean fluorescent intensity of a-tubulin, KRT5, and SARS-CoV-2 N (nucleocapsid), and the mean intracellular (cytoplasmic and nuclear combined) fluorescence intensity of LSD1 in uninfected or SARS-CoV-2-infected cells. n = 3, >20 cells were analyzed for each biological repeat. Mann–Whitney test: ****P < 0.0001 denote significant differences. o Dot plot quantification of the fluorescence intensity of ACE2 and LSD1 in KRT5+SARS-CoV-2 N+ cells. The PCC was calculated for LSD1 and ACE2 colocalization. n = 3, >20 cells were analyzed for each biological repeat. p qRT-PCR analysis of LSD1, ACE2, and TMPRSS2 mRNA expression in uninfected versus SARS-CoV-2-infected HBECs at 48 hpi. Data are mean ± SEM. (n = 3, unpaired t-test: **P < 0.01).
Fig 2: RCOR1 globally represses transcription of newly synthesized transcripts.a Scheme depicting the strategy used to label nascent transcripts in Corin-treated HT22 cells. b Western blot analysis showing the effect on H3 modifications after treating HT22 cells with Corin. Panels are representative of two independent experiments. c Pseudocolor images showing labeling of nascent transcripts in HT22 cells. Scale bar represents 40 µm. Right box plots showing quantitation of fluorescence intensity of nascent transcripts. Red asterisk indicates p < 0.05 statistical significance compared to DMSO treated cells according to non parametric, two-tailed, Mann–Whitney test. d RCOR1 immunoprecipitation showing co-immunoprecipitated RPB1 levels after treating HT22 cells with Corin. This experiment was performed once. e Scheme depicting the strategy used to analyze RPB1 post-translational modifications after Corin treatment. f Analyses of RPB1 dimethylation under Corin treatment. Panels are representative of two independent experiments. g Analyses of RPB1 acetylation under Corin treatment. Panels are representative of two independent experiments. h RPB1-K7ac western blot after performing RPB1 immunoprecipitation from two different replicates of DMSO or Corin treated HT22 cells. RPB1-S2P was assayed as a control showing the precipitation of RPB1. Panels are representative of three independent experiments. i Subcellular fractionation of HT22 cells treated with DMSO or Corin. Cytosol, Nuclear soluble, and chromatin fractions were analyzed for RPB1-K7ac, RPB1, and RCOR1. Panels are representative of two independent experiments. j Graph depicting the RNA levels of c-Fos, egr2 (genes induced by EGF), and eif4a1 (negative control) when DMSO or Corin-treated HT22 cells are stimulated with EGF or PBS. Upper right corner shows the positive immunostaining of the EGF receptor (EGFR) in HT22 cells. Results are shown as columns depicting the mean plus SEM. Red asterisks indicate **p < 0.001 and *p < 0.05 statistical significance compared to DMSO treated, EGF-stimulated cells by unpaired, two-sided T-tests. This experiment was performed using three biological replicates with two technical replicates each. k Working model. Source data are provided as a Source Data file.
Fig 3: RCOR1 establishes an interaction with RNA POL-II after initiation and before elongation.a Western blot analyses of immunoprecipitation of RCOR1 and co-precipitation of RPB1 and its phosphorylated isoforms from whole cell extracts. II-O: Hyperphosphorylated isoforms. II-A: Hypophosphorylated isoforms. RPB1-NTD CoIP panel is representative of four independent experiments while others are representative of two independent experiments. b Western blot analyses of immunoprecipitation of LSD1 (top) or HDAC1 (bottom) and co-precipitation of RPB1. Experiment performed once. c Western blot analysis of immunoprecipitation of RCOR1 and RNA POL-II from MNased chromatin using 2 different RCOR1 antibody concentrations. Panels are representative of two independent experiments. d HA-Pulldown on extracts derived from empty-vector and HA-RCOR1 overexpressing HEK293 cells. Panels are representative of two independent experiments. e Scheme depicting different stages of eukaryotic RNA POL-II transcription. It highlights the steps that are inhibited by THZ1, Flavopiridol, Cordycepin, and Actinomycin D. CTD: Carboxy-terminal domain. S5Ph: Phosphorylated serine 5. S2Ph: Phosphorylated serine 2. f Western blot analyses of RCOR1 immunoprecipitation and RPB1 co-precipitation under inhibition of transcription at different steps. Panels are representative of three independent experiments. g Proximity ligation assay between RCOR1 and RPB1 under DMSO, THZ1 and Actinomycin D treatments in HT22 cells. Right graph shows the distribution of proximal-ligated spots per cell under each treatment. More than 128 cells from three independent experiments were analyzed per treatment. Statistical significance was checked on Graphpad Prism by confirming normal distribution of data by D’Agostino & Pearson, Shapiro-Wilk and KS normality tests. Data was subjected to an ordinary, unpaired, one-way ANOVA test with 99% confidence intervals, **** means p < 0.01. Source data are provided as a Source Data file.
Fig 4: RCOR1 occupancies are positively correlated with gene expression.a Meta-gene plots of RNA-seq data in K562 cells. Lower diagram shows the heatmap of the signal intensity from RNA-seq data sorted by intensity. b Quartile-based comparison of relative RNA POL-II, H3K9ac, and RCOR1 occupancies on the genes in different quartile groups based on RNA-seq signal intensity. Two different RCOR1 datasets (using different antibodies) were analyzed (labeled as #1 and #2). c and d Gene ontology analysis of RCOR1 clusters I and II, respectively. Bar colors represent different ontology categories, highlighted at the bottom.
Fig 5: Association of RCORs with HCC. (A) Correlation between RCORs and immune infiltration from GSCA database; Expression of immune checkpoint moleculars between differentially expressed groups of RCOR1 (B), RCOR2 (C) and RCOR3 (D) in HCC; (E) Survival analysis between high and low HCC stemness groups; (F) Multivariate cox regression analysis; (G) Stemness indices among different molecular subtypes; (H) Relationship in tumor stage, subtype and stemness status. *p < 0.05, **p < 0.01, and ***p < 0.001.
Supplier Page from Abcam for Anti-CoREST antibody [EPR13825]