Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 expression was elevated in mice after renal IRI. All mice were subjected to ischaemia 30 min and reperfusion 6, 12 and 24 h, respectively. (A) LSD1 mRNA level was elevated after renal IRI. (B‐C) LSD1 protein level was elevated after renal IRI, and the quantification also was shown. (D) The immunohistochemical staining of LSD1 was examined in renal tissues at 6, 12 or 24 h of reperfusion time (×400; scale bars = 40 μm). (E) The morphological changes of the renal tissues detected by H&E staining at 6, 12 or 24 h of reperfusion (×400; scale bars = 40 μm)(F–G)The levels of Cr and BUN were detected after renal IRI ( n = 8) The results were expressed as mean ± standard error of mean (SEM). * p < 0.05, when compared with the sham group. ** p < 0.05, when compared with the IR 6 h group. *** p < 0.05, when compared with the IR 12 h group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 inhibitor protected kidney tissue against IRI in mice. All mice were subjected to ischaemia 30 min and then reperfusion 24 h. (A–B) The effect of LSD1 inhibitor at various concentration (2.5, 5 or 10 mg/kg) on the levels of Cr and BUN in IRI group. (C–F) The effect of LSD1 inhibitor at various concentration on LSD1 and H3K9me2 expression in mice and the quantification. (G) Representative pictures of immunohistochemistry for H3K9me2 (×400; scale bars = 40 μm) ( n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the sham group. # p < 0.05, when compared with the IRI group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Concentration Assay, Expressing, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 inhibitor alleviated ferroptosis and oxidative stress that were caused by renal IRI in mice. All mice were subjected to ischaemia 30 min and then reperfusion 24 h. (A–F) The regulation of LSD1 inhibitor on the expression of ASCL4, 4‐HNE, GPX4 and FSP1 after renal IRI, and quantification was also shown. (G) The regulation of LSD1 inhibitor on the SOD activity in mice subjected to renal IRI. (H) The regulation of LSD1 inhibitor on the MDA content in mice subjected to renal IRI. (I) The regulation of LSD1 inhibitor on the GSH level in mice subjected to renal IRI. (J) The regulation of LSD1 inhibitor on the Fe 2+ level in mice subjected to renal IRI. (K) The effect of TCP at the concentration (10 mg/kg) on renal structure damage detected by H&E staining (×400; scale bars = 40 μm) ( n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the sham group. # p < 0.05, when compared with the IRI group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Expressing, Activity Assay, Concentration Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 expression were elevated during H/R induced injury in vitro. The HK‐2 cells were subjected to hypoxia 12 h and reoxygenation 2, 4 and 6 h. (A) The cell viability was examined by CCK‐8 kit in HK‐2 cells after H/R. (B–C) LSD1 expression was detected by Western blot in HK‐2 cells after H/R, and the quantification was also shown. (D–E) Various concentration of LSD1 inhibitor was employed to examine LSD1 expression, and the quantification was also shown. (F–I) The expression of LSD1 and H3K9me2 was examined by Western blot after inhibition of LSD1 with TCP or si‐RNA against LSD1 ( n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the control group. # p < 0.05, when compared with the H/R group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Expressing, In Vitro, CCK-8 Assay, Western Blot, Concentration Assay, Inhibition, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 inhibition decreased TLR4/NOX4, ferroptosis and oxidative stress that were caused by H/R in HK‐2 cells. The HK‐2 cells were subjected to hypoxia 12 h and reoxygenation 6 h. The cells were pretreated with TCP or transfected with si‐NC or si‐LSD1 for 24 h and then subjected to H/R. (A–C) The expression of TLR/NOX4 pathway was examined by Western blot, and the quantification was also shown. (D–I) The regulatory effect of LSD1 on ASCL4, 4‐HNE, GPX4 and FSP1 expression in HK‐2 cells after H/R, and quantification. (J) The regulation of LSD1 on SOD activity in HK‐2 cells after H/R. (K) The regulation of LSD1 on MDA content in HK‐2 cells after H/R. (L) The regulation of LSD1 on GSH level in HK‐2 cells after H/R. (M) The regulation of LSD1 on Fe 2+ level in HK‐2 cells after H/R ( n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the control group. # p < 0.05, when compared with the H/R + DMSO group. △ p < 0.05, when compared with the H/R + si‐NC group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Inhibition, Transfection, Expressing, Western Blot, Activity Assay, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 aggravates ferroptosis and oxidative stress induced by H/R process via activation of TLR4/NOX4 pathway in HK‐2 cells. The HK‐2 cells were subjected to hypoxia 12 h and reoxygenation 6 h. The HK‐2 cells were transfected with si‐NC or LSD1 for 24 h, and then subjected to H/R, with or without infected with adenovirus carrying TLR4. (A–D) The expression of LSD1 and H3K9me2 was examined by Western blot after LSD1 silence with or without ad‐TLR4, and quantification was also shown. (E‐G) The expression of TLR4/NOX4 was examined by Western blot after LSD1 silence with or without ad‐TLR4, and quantification was also shown. (H‐M) The expression of ASCL4, 4‐HNE, GPX4 and FSP1 was examined by Western blot after LSD1 silence with or without ad‐TLR4, and quantification was also shown. (N‐Q) The SOD, MDA, GSH and Fe2+ levels were detected after LSD1 silence with or without ad‐TLR4, and quantification was also shown (n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the control group. #P < 0.05, when compared with the H/R + si‐NC group. △ p < 0.05, when compared with the H/R + si‐LSD1 group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Activation Assay, Transfection, Infection, Expressing, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 activated TLR4 transcriptional activity of TLR4 through removal of H3K9me2 from TLR4 promoter. (A) Promoter sequence of TLR4 (from −2000 bp to +600 bp) was obtained from NCBI public database. (B) HK2 cells were transfected with indicated plasmids carrying Flag‐LSD1 and truncation of TLR4 promoter, and transcriptional activity of TLR4 promoter was detected by luciferase reporter assay. * p < 0.05, when compared with the Flag group. (C) The binding of H3K9me2 to TLR4 promoter was tested by ChIP assay. * p < 0.05, when compared with the con group. (D) mRNA level of TLR4 was assessed by qPCR. * p < 0.05, when compared with the con group. (E) ChIP assay was employed to evaluated the enrichment of H3K9me2 in TLR4 promoter. * p < 0.05, when compared with the con group. ** p < 0.05, when compared with the H/R + DMSO group. (F) mRNA level of TLR4 was assessed by qPCR. * p < 0.05, when compared with the con group. ** p < 0.05, when compared with the H/R + DMSO group. (G) The binding of H3K9me2 to TLR4 promoter was tested by ChIP assay. * p < 0.05, when compared with the con group. ** p < 0.05, when compared with the H/R + NC group. (H) mRNA level of TLR4 was assessed by qPCR. * p < 0.05, when compared with the con group. ** p < 0.05, when compared with the H/R + NC group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Activity Assay, Sequencing, Transfection, Luciferase, Reporter Assay, Binding Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Lysine‐specific demethylase 1 aggravated oxidative stress and ferroptosis induced by renal ischemia and reperfusion injury through activation of TLR4 / NOX4 pathway in mice

doi: 10.1111/jcmm.17444

Figure Lengend Snippet: LSD1 inhibitor ameliorated TLR4/NOX4 activation that was induced by renal IRI in mice. All mice were subjected to ischaemia 30 min and then reperfusion 24 h. (A–C) The regulation of LSD1 inhibitor on TLR4/NOX4 expression in mice after IRI, and quantification was also shown. (D–E) Representative pictures of immunohistochemistry for TLR4 and NOX4 (×400; scale bars = 40 μm). ( n = 8). The results were expressed as mean ± SEM. * p < 0.05, when compared with the sham group. # p < 0.05, when compared with the IRI group

Article Snippet: To study the effects of LSD1, TCP (MedChemExpress) was injected intraperitoneally at different doses (2.5, 5, 10 mg/kg) before IRI model establishment, once a day for 1 week.

Techniques: Activation Assay, Expressing, Immunohistochemistry

Journal: bioRxiv

Article Title: Cancer-associated KBTBD4 mutations induce differentiation defects and confer a unique therapeutic vulnerability

doi: 10.64898/2026.03.12.711277

Figure Lengend Snippet: (A) Volcano plot showing global protein expression in HL60 cells transduced with P311PP mutant. Blue and red dots show significantly decreased or increased proteins (adjusted p-value<0.05). CoREST components RREB1, RCOR1, KDM1A, RCOR3, and ZNF217 are highlighted in the volcano plot. The global proteome data was plotted after excluding PCDHGA11. (B) High throughput compound screening in HL60 cells stably expressing RCOR1-GFP treated with UM171 (200nM). Schematic of the screening rationale (top panel). The compounds were added at a final concentration of 1µM, and flow analysis was performed after 3 hours post treatment to measure the relative GFP expression (bottom panel). Red dots show the hits from the screen and the table on right-side shows the functional classification of the hits. (C) A dose-titration experiment showing the rescue of RCOR1-GFP by two class I specific HDAC inhibitors (mocetinostat and romidepsin) and three Pan HDAC inhibitors (belinostat, pracinostat and vorinostat). GFP mean fluorescence intensity of DMSO treatment was used as controls. Data from 3 replicates from 1 of 2 independent experiments with similar results are shown. (D) RCOR1 protein levels in P311PP expressing HL60 cells either treated with DMSO, belinostat (320nM) or mocetinostat (300nM) for 3 hours. (E) RCOR1 ELM2-GFP clones were expressed in HL60 cells and analyzed for the interaction with HDAC2 through immunoprecipitation. The degradation profiles of the corresponding alanine substitution clones to UM171 treatment are represented as a heat map. (F) Schematic representation of the mechanistic basis of HDAC inhibitors in preventing the CoREST degradation.

Article Snippet: The proteins were transferred to iBlot 2 PVDF membranes (#IB24001, Thermo Fisher Scientific) and probed with the following primary antibodies: RCOR1 (#14567), KDM1A (LSD1, #2184S), HDAC2 (#5113T, all from Cell Signaling Technology), GFP (#ab290, Abcam), and Actin (#612656, BD Biosciences).

Techniques: Expressing, Transduction, Mutagenesis, High Throughput Screening Assay, Stable Transfection, Concentration Assay, Functional Assay, Titration, Fluorescence, Clone Assay, Immunoprecipitation

Journal: NPJ Precision Oncology

Article Title: LSD1 deficiency in breast cancer cells promotes the formation of pre-metastatic niches

doi: 10.1038/s41698-024-00751-2

Figure Lengend Snippet: a Western blot analysis of LSD1 expression in MCF7 cells transfected with the control construct (Control), MCF7 cells transfected with LSD1 shRNA (LSD1 KD), and LSD1 KD MCF7 cells in which LSD1 expression is restored (Rescue). b qRT-PCR analysis of LSD1 expression in Control, LSD1 KD, and Rescue cells. Data are presented as means ± SEM ( n = 3). *** p < 0.001. c Western blot analysis of the exosomal markers CD63 and TSG101 in purified exosomes from Control, LSD1 KD, and Rescue cells. d Transmission electron micrographs of exosomes from Control, LSD1 KD, and Rescue cells. Scale bars: 100 nm. e Nanoparticle tracking analysis (NTA) of the diameter and concentration of exosomes from Control, LSD1 KD, and Rescue cells. f Schematic representation (created with BioRender.com) of exosome injection via tail vein and the intracardiac injection of luciferase-labeled MCF7 cells into BALB/c-nu/nu mice. g , h Bioluminescence imaging of the whole body and lungs of mice injected with luciferase-labeled MCF7 cells and treated with the indicated exosomes. i The tumor burden in lungs was quantified by measuring the total flux (photons/s) in bioluminescence imaging. Data are presented as means ± SEM ( n = 5). * p < 0.05. j Immunohistochemical (IHC) staining for Ki67 in lung sections from mice treated with the indicated exosomes.

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature for 30 min, and then incubated at 4 °C overnight with primary antibodies targeting occludin (ABclonal, A2601, 1:1000), ZO-1 (Proteintech, 21773-1-AP, 1:500), LSD1 (Cell Signaling Technology, 2139S, 1:1000), GAPDH (Bioworld, AP0063, 1:2000), CD63 (Abcam, ab59479, 1:1000), and TSG101 (Abcam, ab83, 1:500).

Techniques: Western Blot, Expressing, Transfection, Control, Construct, shRNA, Quantitative RT-PCR, Purification, Transmission Assay, Concentration Assay, Injection, Luciferase, Labeling, Imaging, Immunohistochemical staining, Immunohistochemistry

Journal: NPJ Precision Oncology

Article Title: LSD1 deficiency in breast cancer cells promotes the formation of pre-metastatic niches

doi: 10.1038/s41698-024-00751-2

Figure Lengend Snippet: a Western blot analysis of LSD1 expression in Control, LSD1 knockdown (KD), and Rescue cells in three independent batches of samples. b Venn diagram showing the number of differentially expressed miRNAs between Control and LSD1 KD exosomes and between LSD1 KD and Rescue exosomes. c Heat map of the exosomal miRNAs that are significantly upregulated in LSD1 KD exosomes and restored in Rescue exosomes. d qRT-PCR analysis of the expression levels of the indicated miRNAs in Control, LSD1 KD, and Rescue exosomes. Data are presented as means ± SEM ( n = 3 unless otherwise indicated). * p < 0.05, ** p < 0.01.

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature for 30 min, and then incubated at 4 °C overnight with primary antibodies targeting occludin (ABclonal, A2601, 1:1000), ZO-1 (Proteintech, 21773-1-AP, 1:500), LSD1 (Cell Signaling Technology, 2139S, 1:1000), GAPDH (Bioworld, AP0063, 1:2000), CD63 (Abcam, ab59479, 1:1000), and TSG101 (Abcam, ab83, 1:500).

Techniques: Western Blot, Expressing, Control, Knockdown, Quantitative RT-PCR

Journal: NPJ Precision Oncology

Article Title: LSD1 deficiency in breast cancer cells promotes the formation of pre-metastatic niches

doi: 10.1038/s41698-024-00751-2

Figure Lengend Snippet: a Venn diagram showing the number of differentially expressed circRNAs between Control and LSD1 knockdown (KD) cells and between LSD1 KD and Rescue cells. b Heat map of the circRNAs that are both significantly changed in LSD1 KD cells and restored in Rescue cells. c The relative expression of circDOCK1 and circSEMA3A in Control, LSD1 KD, and Rescue cells ( n = 4). d Venn diagram showing the number of circDOCK1 target miRNAs predicted by circBank and Circinteractome and the number of miRNAs upregulated in LSD1 KD exosomes. e Schematic diagram showing the binding sites of miR-1270, miR-1246, and miR-1265 in circDOCK1. f PCR analysis of circDOCK1 and its linear isoform DOCK1 after treatment with RNase R. g qRT-PCR analysis of circDOCK1 and its linear DOCK1 after treatment with RNase R. h Schematic diagram showing the genomic locus of the DOCK1 gene and the back-splicing of circDOCK1. The red arrow indicates the back-splicing site of DOCK1, which was confirmed by Sanger sequencing. i qRT-PCR analysis of the nuclear-cytoplasmic fractionation of circDOCK1. j Fluorescence in situ hybridization (FISH) analysis of the subcellular localization of circDOCK1 in MCF7 cells. Data are presented as means ± SEM ( n = 3 unless otherwise indicated). *** p < 0.001.

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature for 30 min, and then incubated at 4 °C overnight with primary antibodies targeting occludin (ABclonal, A2601, 1:1000), ZO-1 (Proteintech, 21773-1-AP, 1:500), LSD1 (Cell Signaling Technology, 2139S, 1:1000), GAPDH (Bioworld, AP0063, 1:2000), CD63 (Abcam, ab59479, 1:1000), and TSG101 (Abcam, ab83, 1:500).

Techniques: Control, Knockdown, Expressing, Binding Assay, Quantitative RT-PCR, Sequencing, Fractionation, Fluorescence, In Situ Hybridization

Journal: NPJ Precision Oncology

Article Title: LSD1 deficiency in breast cancer cells promotes the formation of pre-metastatic niches

doi: 10.1038/s41698-024-00751-2

Figure Lengend Snippet: In breast cancer cells, LSD1 deficiency decreases the expression of circDOCK1, resulting in increased levels of miR-1270 in exosomes. Vascular endothelial cells in distant organs uptake these breast cancer cell-derived exosomes with increased miR-1270 expression, leading to the downregulation of the expression of ZO-1 and other tight junction proteins. Vascular permeability is subsequently enhanced, leading to the formation of pre-metastatic niches and the promotion of lung metastasis. Created with BioRender.com.

Article Snippet: The membrane was blocked with 5% nonfat milk at room temperature for 30 min, and then incubated at 4 °C overnight with primary antibodies targeting occludin (ABclonal, A2601, 1:1000), ZO-1 (Proteintech, 21773-1-AP, 1:500), LSD1 (Cell Signaling Technology, 2139S, 1:1000), GAPDH (Bioworld, AP0063, 1:2000), CD63 (Abcam, ab59479, 1:1000), and TSG101 (Abcam, ab83, 1:500).

Techniques: Expressing, Derivative Assay, Permeability

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 1. LSD1 regulates phosphorylation of AKT in colorectal cancer. A, Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for LSD1 across different TCGA datasets. Boxlimitsare set at the third and first quartilerange with central line at the median, withwhiskers depicting 1.5 times the interquartile range. Data points outside this range are represented at outliers (black dots). Black and blue outline indicates data for WT and PIK3CA- mutant tumors, respectively. Red and purple fill represent significant increase and decrease in LSD1 expression, respectively, between PIK3CA-mutant and PIK3CA WT tumors. The numbers under the box plots are the number of samples used to generate the plots. Western blot analysis of empty vector (shEV) or LSD1 KD in SW480 (B) or HT29 (C) cells. Arrowhead indicates correct position of pT308-AKT band. Western blot quantified by densitometric analysis and normalized to b-actin and shEV. Results are represented as mean SD (n ¼ 3). Significance was determined by two-tailed Student t test. LSD1 CRISPR KO clones with or without LSD1 OE plasmid (HA-LSD1) in SW480 (D) or HT29 (E) cells analyzed by Western blot analysis. F, EV, LSD1 KD, or LSD1 OE cells treated with 250 mmol/L H2O2 for 1 hour. G, Brightfield and immunofluorescence images of EV or LSD1 KD HT29 cells under untreated or H2O2-treated conditions. A field was selected in the H2O2-treated shLSD1 cells to facilitate direct comparison of LSD1-deficient and LSD1-proficient cells. White arrow indicates cells with LSD1 expression and orange arrow indicates cells deficient in LSD1 (, P < 0.001; , P < 0.0001; ns, not significant).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Phospho-proteomics, Whisker Assay, Expressing, Mutagenesis, Western Blot, Plasmid Preparation, Two Tailed Test, CRISPR, Clone Assay, Comparison

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 2. LSD1 catalytic activity is dispensable for regulation of gene expression and activation of AKT. A, Metageneplot and heatmap depicting ChIP-seq of LSD1 in WT (n ¼ 2) or LSD1 KO (n ¼ 1) SW480 cells at gene enrichment sites genome-wide. Average plots and heatmaps depicting: LSD1 enrichment peak overlap with DNase-seq peaks in SW480 (B) and H3K4me2 ChIP-seq signal at TSS enrichment sites genome-wide in shEV and shLSD1 SW480 cells (N ¼ 3; C). A–C, Values are derived from CPM (counts per million) normalized reads. E, Differentially expressed genes (DEG) from RNA-seq (log2FC 1 and FDR 0.05 ¼ purple) after 40 nmol/L GSK-LSD1 for 48 hours versus DMSO or shLSD1 versus shEV in SW480 cells (N ¼ 3). D, ChIP-seq gene tracks of representative DEGs in LSD1 versus EV KD SW480 cells with or without LSD1 promoter enrichment. F, Cells pretreated with DMSO or 40 nmol/L GSK-LSD1 for 48 hours then treated with 250 mmol/L H2O2 for 1 hour. Western blots were quantified by densitometric analysis and normalized to loading control and DMSO. Graph represents mean SD; ns, not significant. Significance determined using one-way ANOVA with Tukey multiple comparisons test (N ¼ 3). G, Mixed population LSD1 KO cells were transfected with vector control, HA-LSD1, or HA-LSD1 (K661A) for 48 hours. Whole-cell extract from untreated and cells treated with 250 mmol/L H2O2 for 1 hour were analyzed by Western blot analysis.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Activity Assay, Gene Expression, Activation Assay, ChIP-sequencing, Genome Wide, Derivative Assay, RNA Sequencing, Western Blot, Control, Transfection, Plasmid Preparation

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 3. LSD1 regulates AKT activation via scaffolding of the CoREST complex on chromatin. A, Chromatin affinity assay performed in shEV or shLSD1 with whole-cell extract (WCE) or chromatin-bound fraction. Western blots were quantified by densitometric analysis and normalized to loading control and shEV fraction. Significance determined by two-way ANOVA with Sidak multiple comparisons test (n ¼ 3). Graphs depict mean SD (, Padj < 0.01; , Padj < 0.0001; ns, not significant). B, shEV or shRCOR1 cells treated with 250 mmol/L H2O2 for 1 hour and analyzed by Western blot analysis. C, shEV or shHDAC1 cells treated as in B. D, Model for corin inhibitor mode of action. E, Cells treated with DMSO or 3, 5, or 7 mmol/L corin over time course and analyzed by Western blot analysis. F, Box and whisker plot of fragments per kilobase of transcript per million mapped reads (FPKM) expression values for RCOR1 across different TCGA datasets. Box limits are set at the third and first quartile range with central line at the median, with whiskers depicting 1.5 times the interquartile range. Data points outside this range are represented as outliers (black dots). Black and blue outline indicates data for WT and PIK3CA-mutant tumors, respectively. Red fill represent significant increase in LSD1 expression, respectively, between PIK3CA-mutant and PIK3CA WT tumors. G, Fraction of patients with PIK3CA mutation separated by high expression of LSD1 or RCOR1 versus low expression of both LSD1 and RCOR1. Plotted below is bootstrapped 90% confidence interval for the mean difference in fraction for patients with the PIK3CA mutation between the two groups. Significance determined by permutation test (, P < 0.001).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Activation Assay, Scaffolding, Western Blot, Control, Whisker Assay, Expressing, Mutagenesis

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 4. Gastrointestinal cell lines with mutant PIK3CA are sensitive to LSD1 KD. A, SW480, LoVo, HT29, AGS, HCT116, and RKO cell growth over a 5-day time course determined by the CellTiter-Glo Luminescent Cell Viability Assay (n ¼ 4). Graph depicts mean þ SD. Statistical analyses are performed using two-way ANOVA and Sidak multiple comparisons test with all statistically significant comparisons shown. , Padj < 0.01; , Padj < 0.0001. B, Correlation plot of RNA-seq data from LSD1 versus EV KD in SW480 and HT29 cells. Significant data points are defined as abs(Log2FC) 1 and FDR 0.05 for LSD1 compared with EV KD for each cell line, including those unique to HT29 (purple), unique to SW480 (red), and shared between HT29 and SW480 (black; n ¼ 3). C, Ridge plot depicts LSD1 versus EV KD expression changes of genes contributing to max enrichment score of Hallmark and curated gene sets accessed from the molecular Signatures Database (v6.2). GSEA Ratio shows the ratio of genes contributing to max enrichment score, tothe total number of genes in the gene set. Heatmap on right shows Log2FC value for each gene enriched in HALLMARK_PI3K_AKT_MTOR_SIGNALING in either HT29 or SW480 cells sorted by HT29 Log2FC. D, Network analysis of uniquely upregulated genes in HT29 after LSD1 KD with significantly enriched processes from the Reactome database. Similar terms were manually grouped and size and color of circles were set to indicate number of genes and the P value, respectively. E, Clonogenic growth assay. Significance determined by two-way ANOVA with Sidak multiple comparisons test (n ¼ 3). Results are represented as mean SD (, Padj < 0.01; ns, not significant).

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Mutagenesis, Cell Viability Assay, RNA Sequencing, Expressing, Growth Assay

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 5. LSD1 regulates Snail stability via AKT in PIK3CA C2 domain–mutant cancer cells. Western blot analysis of HT29 (A) or AGS cells treated with DMSO or 10 mmol/L GSK690693 for 48 hours (B). C, Real-time PCR analysis of SNAI1 RNA expression levels after 48-hour DMSO or 10 mmol/L GSK690693 treatment in HT29 and AGS cells. Expression was normalized to Gapdh and DMSO. Results are represented as mean SD. Significance determined by two-way ANOVA with Sidak multiple comparisons test. ns, not significant. D, Proportion of total PIK3CA mutations occurring in the different domains indicated across various cancer types in the TCGA pancancer datasets. E, shEV and shLSD1 HT29 cells treated with DMSO or 10 mmol/L MG-132 for 4 hours and analyzed by Western blot analysis. F, Real-time PCR analysis of LSD1 and SNAI1 RNA expression levels in shEV and shLSD1 HT29 cells as in C. , Padj < 0.0001. G, HT29 shLSD1 cells were transfected with HA-LSD1 alone or in combination with 10 mmol/L GSK690693.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Mutagenesis, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Expressing, Transfection

Journal: Molecular Cancer Research

Article Title: Lysine-Specific Demethylase 1 Mediates AKT Activity and Promotes Epithelial-to-Mesenchymal Transition in PIK3CA-Mutant Colorectal Cancer

doi: 10.1158/1541-7786.mcr-19-0748

Figure Lengend Snippet: Figure 6. LSD1 is required for EGF-induced migration of cells with an active AKT–GSK3b–Snail axis. A, 10 Brightfield images of crystal violet–stained HT29 cells after 48-hour migration through transwell insert. WT, LSD1 KO, 10 mmol/L GSK690693, or 3 mmol/L corin cells were cotreated with 100 ng/mL EGF for 48 hours. B, Quantification of migration normalized to migration counts for untreated cells. Results are represented as mean SD. Significance was determined by one-way ANOVA with Tukey multiple comparisons test. All significant comparisons are shown. , Padj < 0.0001. C, shEV or shLSD1 cells were treated with 100 ng/mL EGF for 24 or 48 hours and analyzed by Western blot. D, Model depicting CoREST complex in the regulation of AKT.

Article Snippet: CRISPR/Cas9 LSD1 KO plasmid (sc-430289) and LSD1 HDR plasmid (sc-430289-HDR) were purchased from Santa Cruz Biotechnology and knockout was performed according to manufacturer's protocol.

Techniques: Migration, Staining, Western Blot

Journal:

Article Title: Gene-expression signature of benign monoclonal gammopathy evident in multiple myeloma is linked to good prognosis

doi: 10.1182/blood-2006-07-037077

Figure Lengend Snippet: Fifty-two SAM-defined genes are differentially expressed in NPC, MGUS, and MM

Article Snippet: 2.05 1.55 3.18 1.36 212038_s_at VDAC1 Ion channel for cytochrome c 2.62 1.56 3.17 1.42 208308_s_at GPI Energy metabolism 1.97 1.48 3.17 1.48 201013_s_at PA1CS DNA synthesis 2.81 1.43 3.17 1.45 202708_s_at HIST2H2BE Chromosome organization and biogenesis 2.35 1.71 3.14 2.11 215071_s_at HIST1H2AC Chromosome organization and biogenesis 3.47 3.62 3.13 1.97 225361_x_at LOC159090 Unknown 3.43 1.68 3.11 1.53 219366_at AVEN Antiapoptosis 2.63 1.76 3.10 1.47 209398_at H1ST1H1C Chromosome organization and biogenesis 5.39 5.53 3.09 2.02 221652_s_at C12orf11 Unknown; sarcoma antigen NY-SAR-95 2.07 1.37 3.06 1.57 225028_at LOC550643 Unknown 3.83 2.01 3.03 1.45 214214_s_at C1QBP Immunity 2.64 1.45 3.03 1.47 201577_at NME1 Nucleotide biosynthesis 3.57 1.62 2.99 1.58 218280_x_at H1ST2H2AA Chromosome organization and biogenesis 4.24 4.76 2.95 1.92 201479_at DKC1 Telomere maintenance 2.24 1.42 2.92 1.43 208864_s_at TXN Redox reactions 2.90 1.68 2.91 1.51 212297_at ATP13A3 Cation transport 2.64 1.42 2.88 1.54 222825_at OTUD6B Unknown 2.66 1.35 2.88 1.40 209267_s_at SLC39A8 Ion transport 3.41 1.86 2.88 1.56 217898_at C15orf24 Unknown 2.35 1.70 2.83 1.33 210275_s_at ZA20D2 Unknown 2.27 1.36 2.81 1.37 213485_s_at ABCC10 ATP-dependent efflux pump; multidrug resistance pump 3.17 1.51 2.76 1.34 200994_at IPO7 Nuclear trafficking 2.08 1.46 2.72 1.37 222428_s_at LARS Protein synthesis 3.64 1.68 2.71 1.32 202591_s_at SSBP1 Mitochondrial DNA replication 2.22 1.42 2.69 1.35 204244_s_at ASK Cell cycle 3.25 1.77 2.67 1.37 225916_at ZNF131 Gene transcription 2.47 1.39 2.63 1.36 202396_at TCERG1 Gene transcription 3.85 1.62 2.63 1.32 213340_s_at K1AA0495 Unknown −2.40 0.78 −2.52 0.75 206150_at TNFRSF7 Immune cell signaling −3.82 0.55 −2.56 0.75 228139_at RIPK3 Cell signaling −2.63 0.74 −2.59 0.77 220066_at CARD15 Antiapoptosis −4.02 0.50 −2.66 0.62 210347_s_at BCL11A Gene transcription 3.48 2.04 −2.67 0.64 232511_at RANBP2L1 Nuclear import 2.07 1.54 −2.71 0.64 214041_x_at RPL37A Ribosomal protein 2.22 1.80 −3.09 0.60 219371_s_at KLF2 Gene transcription 3.81 1.78 −3.11 0.66 202724_s_at FOXO1A Gene transcription 2.49 1.55 −3.27 0.64 215671_at PDE4B Drug metabolism 2.07 1.53 −3.30 0.59 219675_s_at UXS1 Glycosaminoglycan biosynthesis −2.33 0.76 2.97 1.35 Open in a separate window Genes are ordered based on the SAM score in the MM vs MGUS comparison.

Techniques: Binding Assay, DNA Synthesis

Journal: Nature Communications

Article Title: Mitochondrial-cytochrome c oxidase II promotes glutaminolysis to sustain tumor cell survival upon glucose deprivation

doi: 10.1038/s41467-024-55768-9

Figure Lengend Snippet: a – c Indicated cells were subjected to RNA-seq ( a , n = 2 biologically independent samples per experiment), qPCR ( b , n = 3 independent experiments), or western blot ( c ) analyses. d , e H1299-shMT-CO2 cells were transfected with c-JUN for 48 h, followed by western blot ( d ) or qPCR ( e , n = 3 independent experiments) analyses. The samples derive from the same experiment but different gels for GLS1, MT-CO2, GAPDH, and another for c-JUN were processed in parallel ( c , d ). f – i Indicated cells were subjected to measurement of the oxygen consumption rate (OCR) ( f , h ) or complex (II, III, IV) activity ( g , i ) ( n = 3 independent experiments). j Indicated cells were subjected to examine cellular FAD levels ( n = 3 independent experiments). k H1299 cells were treated with an indicated concentration of FAD for 48 h, followed by western blot analyses. l – o Indicated cells were treated with an indicated concentration FAD or riboflavin for 48 h, followed by examining the cellular FAD levels ( l , n , n = 3 independent experiments) or western blot analyses ( m , o ). The samples derive from the same experiment but different gels for GLS1, H3K9me2, GAPDH, and another for c-JUN, MT-CO2 were processed in parallel ( m , o ). p H1299 cells were treated with an indicated concentration of GSK-LSD1 for 36 h, followed by western blot analyses. The samples derive from the same experiment but different gels for GLS1, H3K9me2, GAPDH, and another for c-JUN, H3K4me2 were processed in parallel ( k , p ). q Indicated cells were subjected to western blot analyses. The samples derive from the same experiment but different gels for LSD1, H3K9me2, GAPDH, another for EZH2, H3K27me3, another for MT-CO2, and another for H3K4me2 were processed in parallel. r , s H1299 cells were treated with 20 μM GSK-LSD1 for 36 h ( r ) or H1299-shMT-CO2 cells were treated with 20 μM FAD for 48 h ( s ), followed by CHIP-qPCR analyses ( n = 3 independent experiments). These experiments have been repeated three times with similar results ( c , d , k , m , o – q ). Data were presented as mean ± SD ( b , e , f – j , l , n , r , s ). Comparisons were performed with one-way ANOVA with Tukey’s test ( b , e , j , l , n ) and unpaired two-tailed Student’s t test ( g , i , r , s ).

Article Snippet: Berberine (HY-18258), carboxin (HY-B2064), antimycin A (HY-107406), flavin adenine dinucleotide (FAD+, HY-B1654A), riboflavin (HY-B0456), CB-839 (HY-12248), CP-91149 (HY-13525), etomoxir (HY-50202), and GSK-LSD1 (HY-100546A) were purchased from MCE (Monmouth Junction, NJ, USA).

Techniques: RNA Sequencing, Western Blot, Transfection, Activity Assay, Concentration Assay, ChIP-qPCR, Two Tailed Test