Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity

doi: 10.3164/jcbn.20-9

Figure Lengend Snippet: (A) FAD-dependent demethylation mechanism of methylated lysine by LSD1. (B) Our strategy to detect LSD1 activity through β-elimination.

Article Snippet: It was performed using a LSD1 fluorescent assay kit (Enzo Life Sciences, Inc., BML-AK544-0001).

Techniques: Methylation, Activity Assay

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity

doi: 10.3164/jcbn.20-9

Figure Lengend Snippet: Synthesis of various LSD1 artificial substrates.

Article Snippet: It was performed using a LSD1 fluorescent assay kit (Enzo Life Sciences, Inc., BML-AK544-0001).

Techniques:

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity

doi: 10.3164/jcbn.20-9

Figure Lengend Snippet: Enzymatic reaction of 9a with active LSD1 or heat-denatured LSD1 (90°C for 5 min). Enzymatic reactions were performed in LSD1/HRP buffer, containing 25 µM 9a, 5 ng/µl LSD1 (active or denatured). Absorption was measured with ARVO X5 (filters; 405/10 nm) every 5 min for 3 h at 25°C. The results are shown as mean ± SD. ( n = 3). (A) Time-dependent curve (B) Absorption of 9a after enzymatic reaction for 180 min. * p <0.0001 (Student’s t test).

Article Snippet: It was performed using a LSD1 fluorescent assay kit (Enzo Life Sciences, Inc., BML-AK544-0001).

Techniques:

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity

doi: 10.3164/jcbn.20-9

Figure Lengend Snippet: Detection of H 2 O 2 in HRP assay. A mixture of CeLLestialTM Red, HRP, 9a (25 µM), and LSD1 (0.5 µg/well) were deposited in all wells. The assay plate was incubated at 25°C for 3 h. Absorption was measured with ARVO X5 (filters; Ex. = 531/25 nm, Em. 595/60 nm) every 3 min for 3 h. Experiments were run in triplicate.

Article Snippet: It was performed using a LSD1 fluorescent assay kit (Enzo Life Sciences, Inc., BML-AK544-0001).

Techniques: Incubation

Journal: Journal of Clinical Biochemistry and Nutrition

Article Title: Synthesis of artificial substrate based on inhibitor for detecting LSD1 activity

doi: 10.3164/jcbn.20-9

Figure Lengend Snippet: Enzymatic reaction of 9a with LSD1 in the presence or absence of LSD1 inhibitor. Enzymatic reactions were performed in LSD1/HRP buffer, containing 25 µM 9a, 5 ng/µl LSD1 after pre-incubation in the presence or absence of 160 nM GSK-LSD1 for 30 min. Absorption was measured with ARVO X5 (filters; 405/10 nm) after 3 h incubation at 25°C. The results are shown as mean ± SD ( n = 3). * p <0.001, ** p <0.0001 (Bonferroni-type multiple t test).

Article Snippet: It was performed using a LSD1 fluorescent assay kit (Enzo Life Sciences, Inc., BML-AK544-0001).

Techniques: Incubation

Journal: Nature Communications

Article Title: Caloric restriction leads to druggable LSD1-dependent cancer stem cells expansion

doi: 10.1038/s41467-023-44348-y

Figure Lengend Snippet: a – f Percentage of apoptotic and dead cells in different experimental conditions: pharmacologically inhibited ( a ) or genetically depleted ( b ) LSD1 in NB4 cells, ±OSI-906 (5 μM) for 48 h followed by addition of TRAIL (100 ng/ml) for 20 h, ( n = 4 biological replicates, representative of 4 independent experiments); c Percentage of apoptotic/dead cells (by Annexin V and PI staining) of LSD1i- (2.5 μM; 24 h) and/or OSI-906 (10 μM; 24 h) treated or untreated NPM1c+ blasts, ±TRAIL (100 ng/ml) for 20 h ( n = 3 biological replicates, representative of 3 independent experiments). d LSD1 WT and KO NB4 cells treated with TRAIL and OSI-906 (5 μM), PI3K inhibitor Ly294002 (10 μM) and ERK inhibitor Trametinib (4 nM), alone or in combination, ( n = 3 biological replicates, representative of 3 independent experiments); e LSD1 WT or KO cells, with shRNASEL or control, ±OSI-906 ( n = 4 biological replicates, representative of 3 independent experiments); f LSD1 WT or KO cells, ±TRAIL or OSI-906 and different pan- or specific caspase inhibitors ( n = 3 biological replicates, representative of 4 independent experiments). g , h CFLAR expression on APL blasts from mice exposed to SD or CR diet ±LSD1i, by WB ( g ) or FACS (SD, n = 3; CR, n = 4; SD+LSD1i = 3; CR+LSD1i, n = 6) (“ n ” number of mice) ( h ). MFI: Mean Fluorescence Intensity. i , j CFLAR expression in NB4 cells ±LSD1i ±OSI-906 ( i ) and NB4 LSD1 WT/KO cells ±OSI-906 ( j ), ±TRAIL for indicated time by WB. Note that the CFLAR migration appears delayed in NB4 LSDKO cells on the gradient gel, suggesting modification upon LSD1 ablation; k – l Effect of CFLAR overexpression on TRAIL-induced death of NB4 LSD1 WT or KO cells ±OSI-906, by annexin/PI staining ( k , n = 4 biological replicates, representative of 3 independent experiments) and cleaved caspase 3 staining ( l , n = 3 biological replicates, representative of 2 independent experiments). Data are expressed as mean ±SD, two-way ANOVA with post-hoc Tukey’s multiple comparison. Western blots are representative of 3 independent experiments. Source data are provided as a Source Data file.

Article Snippet: The antibodies used for flow cytometry are CD45.2 monoclonal antibody (104), FITC (eBioscience™; #11-0454-82, dilution 1:100); CD45.2 monoclonal antibody (104), PE (eBioscience™; #12-0454-82, dilution 1:100); CD45.2 monoclonal antibody (104), APC (eBioscience™; #17-0454-82, dilution 1:100); CD45.2 monoclonal antibody (104), APC-Cy7 (BD Pharmingen™; #560694, dilution 1:100); CD45.1 monoclonal antibody (A20), PE-Cyanine7 (eBioscience™; #25-0453-82, dilution 1:100); CD45.1 monoclonal antibody (A20), PE (eBioscience; # 12-0453-82, dilution 1:100); alexa fluor® 488 mouse anti-Ki-67 (B56)(BD Pharmingen™; # 558616, dilution 1:25); alexa fluor® 647 Mouse anti-Ki-67(B56) (BD Pharmingen™; #561126, dilution 1:25); PE rabbit anti- active caspase-3 (C92-605) (BD Pharmingen™; # 550821; dilution 1:10); CFLAR/FLIP (D5J1E) rabbit mAb (Cell signaling; #56343, dilution 1:100), CD177 antibody (R&D Systems; #MAB8186, dilution 1:150), CD34 monoclonal antibody (RAM34) FITC (eBioscience™, # 11-0341-82, dilution 1:100), FcyRIII/II (CD16/CD32) monoclonal antibody (93), eFluor™ 450 (eBioscience™, # 48-0161-82, dilution 1:100), GR1 (Ly-6G/Ly-6C) monoclonal antibody (RB6-8C5), PE, (eBioscience™, # 12-5931-82, dilution 1:100), CD117 (c-Kit) monoclonal antibody (ACK2), APC-eFluor™ 780, (eBioscience™, # 47-1172-82, dilution 1:100), Sca-1 monoclonal antibody (D7), PerCP-Cyanine5.5 (eBioscience™, # 45-5981-82, dilution 1:100), Flk2 (Flt3/CD135) monoclonal antibody (A2F10), APC (eBioscience™, # 17-1351-82, dilution 1:100); CD11b monoclonal antibody (M1/70), FITC, (eBioscience™, #11-0112-82) and anti-KDM1/LSD1 poly clonal antibody (abcam, # ab17721, dilution 1:100).

Techniques: Staining, Expressing, Fluorescence, Migration, Modification, Over Expression, Comparison, Western Blot