Journal: Journal of Clinical Medicine

Article Title: Targeting KDM1A in Neuroblastoma with NCL-1 Induces a Less Aggressive Phenotype and Suppresses Angiogenesis

doi: 10.3390/jcm13206081

Figure Lengend Snippet: NCL-1 efficiently suppresses neuroblastoma cell viability in vitro and inhibits KDM1A-mediated histone demethylation. ( A ). The effective dose of NCL-1 required to inhibit cell viability by 50% (EC 50 ) was calculated for the human neuroblastoma cell lines SH-EP, SK N BE, SK-N-AS, and IMR-5. The EC 50 and the inhibitory concentration (IC 50 ) values were identical. Cell viability was assessed using the MTT assay after treatment with 5–80 µM NCL-1 for 72 h. Treatment with the solvent (DMSO) alone served as the control. ( B ) Whole-cell lysates were prepared in RIPA buffer from cultures treated with 40 µM NCL-1 or DMSO (control) for 24 h and 72 h, then sonicated to disrupt protein complexes. Relative KDM1A activity was analyzed using the EpiQuik™ KDM1A/LSD1 activity/inhibition assay. Significant differences between treatment groups were assessed by Student’s t -test. All comparisons between treatment groups and the corresponding controls had p -values < 0.001. *** p < 0.0001.

Article Snippet: Whole-cell lysates were prepared in RIPA buffer containing 1% ( w/v ) SDS and Complete ® protease inhibitor cocktail (Roche) and analyzed using the EpiQuikTM Histone Demethylase LSD1 Activity/Inhibition Assay Kit (Epigentek, New York, NY, USA) according to the manufacturer’s instructions.

Techniques: In Vitro, Concentration Assay, MTT Assay, Solvent, Control, Sonication, Activity Assay, Inhibition

Journal: Journal of Clinical Medicine

Article Title: Targeting KDM1A in Neuroblastoma with NCL-1 Induces a Less Aggressive Phenotype and Suppresses Angiogenesis

doi: 10.3390/jcm13206081

Figure Lengend Snippet: KDM1A inhibition with NCL-1 mimics functional effects of KDM1A knockdown in vitro. ( A ). Neuroblastoma cell lines were treated for 72 h with 40 µM NCL-1. Cell viability was assessed in MTT assays, cell proliferation in an ELISA (BrdU integration), cell death in the Cell Death ELISA, and NTS expression by qPCR. ( B ). Outgrowing neurites are indicated by black arrowheads in micrographs of cultures treated with 40µM NCL-1 or DMSO (control). Scale bars: 50 µm. ( C ). Cell viability (CellTiter-Glo ® Luminescent Cell Viability Assay) was used to assess effective dose of NCL-1 required to inhibit 50% activity (EC 50 ) in the OHC-NB1 model at 72 h. The EC 50 and the inhibitory concentration (IC 50 ) values were identical. Treatment with DMSO alone served as the control. ( D ). OHC-NB1 model viability and cell death after 72 h NCL-1 treatment relative to controls (DMSO) are presented as bar graphs. ( E ). The effective dose of NCL-1 required to inhibit SK N BE cell viability by 50% (EC 50 ) at 72 h was calculated for treatment with NCL 1 alone or in combination with the indicated retinoic acid (RA) doses. The EC 50 and the inhibitory concentration (IC 50 ) values were identical. The effects of combination treatment with retinoic acid (RA) or NCL 1 alone in SK-N-BE cells on markers of differentiation are shown as bar graphs for relative NTS and MAP2 expression (qPCR) after 24 h ( F ), filamentous cytoskeletal actin by phalloidin staining in representative micrographs after 72 h ( G ) and bar graphs of total numbers of neurite-like structures visible per cell in 125 randomly selected fields after both 24 h and 72 h ( H ). The fold-changes in every treatment group were compared to those in the DMSO control (also without retinoic acid = 1). Significant differences between treatment groups were analyzed using Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Whole-cell lysates were prepared in RIPA buffer containing 1% ( w/v ) SDS and Complete ® protease inhibitor cocktail (Roche) and analyzed using the EpiQuikTM Histone Demethylase LSD1 Activity/Inhibition Assay Kit (Epigentek, New York, NY, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Functional Assay, Knockdown, In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Control, Cell Viability Assay, Activity Assay, Concentration Assay, Staining

Journal: Journal of Clinical Medicine

Article Title: Targeting KDM1A in Neuroblastoma with NCL-1 Induces a Less Aggressive Phenotype and Suppresses Angiogenesis

doi: 10.3390/jcm13206081

Figure Lengend Snippet: Inhibiting KDM1A in HUVEC-C cells in vitro disrupts angiogenic activities. ( A ). Whole-cell lysates were prepared from HUVEC-C and neuroblastoma cell lines in RIPA buffer with sonication to disrupt protein complexes and separated on 15% SDS-PAGE. Basal KDM1A protein expression was detected by Western blotting. HUVEC-C cells were treated with 40 µM NCL-1 or DMSO (control) for 72 h, then relative KDM1A activity was assessed using the EpiQuik™ KDM1A/LSD1 activity/inhibition assay ( B ), cell viability was assessed in MTT assays ( C ), cell proliferation was assessed in an ELISA for BrdU incorporation in DNA ( D ) and cell death was assessed in the Cell Death ELISA ( E , F ). Wound healing was analyzed by scratch assay, with representative pictures of scratch growing after 30 h of treatment. Scale bars: 50 µm. ( G ). Representative pictures are shown of HUVEC-C cells in tube formation assays after 8 h of treatment with NCL-1 or DMSO (control). Scale bars: 100 µm. ( H ). Mean tube length and mesh size were calculated for tubes formed after 24 h in the presence of NCL-1 or DMSO (control). Significant differences between treatment groups were assessed by Student’s t -test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Whole-cell lysates were prepared in RIPA buffer containing 1% ( w/v ) SDS and Complete ® protease inhibitor cocktail (Roche) and analyzed using the EpiQuikTM Histone Demethylase LSD1 Activity/Inhibition Assay Kit (Epigentek, New York, NY, USA) according to the manufacturer’s instructions.

Techniques: In Vitro, Sonication, SDS Page, Expressing, Western Blot, Control, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay, Wound Healing Assay

Journal: Molecules

Article Title: Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation

doi: 10.3390/molecules29112446

Figure Lengend Snippet: Molecular docking results for LSD1. ( A ) The crystal structure of LSD1 in complex with CoREST (PDB ID: 5YJB) is depicted. The FAD co-factor is labelled and predicted ligand-binding sites are coloured brown. 4-[5-(piperidin-4-ylmethoxy)-2-( p -tolyl)pyridin-3-yl]benzonitrile was used as the positive control inhibitor and was docked to the substrate-binding cavity of LSD1. The binding affinity was predicted to be −9.5 kcal/mol. ( B ) The phenolic compounds OLP, OLC, HTA, and HT were screened against the substrate-binding cavity and the binding affinities are provided (kcal/mol). Key residues are labelled, with those predicted to form hydrogen bonds and π–π stacking interactions italicised.

Article Snippet: The inhibitory activity of TCP, HT, HTA, and OLP against purified LSD1 was measured using a commercially available histone demethylase LSD1 inhibitory screening assay core kit (Epigentek P-3075A, Farmingdale, NY, USA) according to the manufacturer’s instructions.

Techniques: Ligand Binding Assay, Positive Control, Binding Assay

Journal: Molecules

Article Title: Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation

doi: 10.3390/molecules29112446

Figure Lengend Snippet: Protein–peptide docking results for histone H3 and LSD1. ( A ) Crystal structure of LSD1-CoREST in complex with the N-terminal residues of the histone H3 peptide. The FAD co-factor is coloured brown. Blind protein–peptide docking was performed to examine the preferential binding site of the histone H3 peptide in the presence of ligands bound to the substrate-binding cavity. The histone H3 peptide was docked to the crystal structure of LSD1-CoREST in the ( B ) absence and ( C ) presence of phenolic compounds bound to the substrate-binding cavity. The results are shown for OLC. ( Aii – Cii ) The protein–peptide interactions were evaluated using PDBePISA. The residues of the LSD1 substrate-binding cavity that were predicted to form salt bridges with R2 and R8 (underlined) of the histone H3 peptide are labelled.

Article Snippet: The inhibitory activity of TCP, HT, HTA, and OLP against purified LSD1 was measured using a commercially available histone demethylase LSD1 inhibitory screening assay core kit (Epigentek P-3075A, Farmingdale, NY, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay

Journal: Molecules

Article Title: Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation

doi: 10.3390/molecules29112446

Figure Lengend Snippet: Inhibitory activity of phenolic compounds against LSD1. ( A ) The results of the control compound TCP and the phenolic compounds HT, HTA, and OLP from the direct enzymatic assays are provided. The data presented denote the mean ± SEM from duplicate (TCP, HTA, and OLP) and triplicate (HT) assays (representative results from n = 3 independent experiments). ( B ) A separate set of experiments were performed by Reaction Biology Corporation using the positive control inhibitor HCl 489479 and the phenolic compounds OLP, HT, HTA, and OLC. The demethylase activity (%) of LSD1 was measured for the control compound HCl 489479 and the phenolic compounds at concentrations ranging from 0–10 μM and 0–100 μM, respectively.

Article Snippet: The inhibitory activity of TCP, HT, HTA, and OLP against purified LSD1 was measured using a commercially available histone demethylase LSD1 inhibitory screening assay core kit (Epigentek P-3075A, Farmingdale, NY, USA) according to the manufacturer’s instructions.

Techniques: Activity Assay, Control, Positive Control

Journal: Cancer Immunology, Immunotherapy

Article Title: Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

doi: 10.1007/s00262-024-03645-1

Figure Lengend Snippet: Role of riboflavin in driving TAMs morphology and polarization. A Schematic overview of the biochemical link between Riboflavin (vitamin B2) and LSD1 enzyme. B At left, riboflavin levels expressed as peak area in S- and L-TAM populations in the 14 TAM pairs. At right, LSD1 activity of S- and L-TAM pairs obtained from 8 CLM-patients expressed as OD. C LSD1 expression analyses in S- and L-TAMs ( n = 4 CLM patients). Gene expressions were normalized using GAPDH as a housekeeping gene. D At left, LSD1 expression levels in M1 and M2 in-vitro polarized monocytes. At right, LSD1 activity of M1 and M2 polarized monocytes expressed as OD. Gene expressions were normalized using GAPDH as a housekeeping gene. Gene expression data are represented as mean ± SD of the average of three biological replicates. * p < 0.05, ** p < 0.01 Wilcoxon Mann–Whitney test

Article Snippet: Lysine-specific demethylase 1 (LSD1) enzyme activity was evaluated using the Abcam KDM1/LSD1 Activity Quantification kit (ab113459, Abcam) according to the manufacturer’s protocol.

Techniques: Activity Assay, Expressing, In Vitro, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy

Article Title: Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

doi: 10.1007/s00262-024-03645-1

Figure Lengend Snippet: Gene analysis on patient-derived tumor cells highlighted high TNFα expression. A Representative immunohistochemistry images showing a CLM patient with higher S-TAMs (left) and higher LTAMs (right) in the peritumoral area. Scale bar: 30 µm. B TNFα and TGFβ expression in primary cells from patients with higher S-TAMs ( n = 3) and with higher L-TAMs ( n = 3). C LSD1 expression in primary cells from patients with higher S-TAMs ( n = 3) and with higher L-TAMs ( n = 3). Gene expressions were normalized using GAPDH as a housekeeping gene. Data are represented as mean ± SD of the average of three biological replicates (Mann–Whitney test; * p < 0.05)

Article Snippet: Lysine-specific demethylase 1 (LSD1) enzyme activity was evaluated using the Abcam KDM1/LSD1 Activity Quantification kit (ab113459, Abcam) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Expressing, Immunohistochemistry, MANN-WHITNEY

Journal: Cancer Immunology, Immunotherapy

Article Title: Riboflavin-LSD1 axis participates in the in vivo tumor-associated macrophage morphology in human colorectal liver metastases

doi: 10.1007/s00262-024-03645-1

Figure Lengend Snippet: LSD1 and riboflavin receptors are increased in M2 macrophages under inflammatory stimuli. A LSD1 expression in monocytes differentiates into M2 macrophages exposed to TNFα (20 ng/ml) for 18 h. B LSD1 expression in monocytes differentiates into M2 macrophages exposed to TGFβ (10 ng/ml) for 18 h. C Riboflavin receptors (SLC52A2 and SLC52A3) expressions in M2 macrophages exposed to TNFα (20 ng/ml) or TGFβ (10 ng/ml) for 18 h. D Riboflavin receptors (SLC52A2 and SLC52A3) expressions in M1, M2, and M2 exposed to TNFα (20 ng/ml) or TGFβ (10 ng/ml) for 18 h. Gene expressions were normalized using GAPDH as a housekeeping gene. Data are represented as mean ± SD of the average of three biological replicates. ** p < 0.01; *** p < 0.001 Mann–Whitney test when comparing two groups. * p < 0.05; ** p < 0.01 One-way ANOVA when comparing three groups

Article Snippet: Lysine-specific demethylase 1 (LSD1) enzyme activity was evaluated using the Abcam KDM1/LSD1 Activity Quantification kit (ab113459, Abcam) according to the manufacturer’s protocol.

Techniques: Expressing, MANN-WHITNEY