Journal: Food & Nutrition Research

Article Title: Arborinine from Glycosmis parva leaf extract inhibits clear-cell renal cell carcinoma by inhibiting KDM1A/UBE2O signaling

doi: 10.29219/fnr.v66.8714

Figure Lengend Snippet: Arborinine inhibits KDM1A in clear-cell renal cell carcinoma (ccRCC). (a) The lowest-energy docking structure of Arborinine with KDM1A (PDB code: 2V1D) generated by molecular docking with residues of KDM1A marked, showing Arborinine buried in a hydrophobic pocket. Western blotting and densitometry showing (b) KDM1A levels in treatment-naïve and resistant ccRCC cell lines; (c) levels H3K4me1/2 and H3K9me1/2 normalized to H3 in response to different doses of Arborinine in 786O cells treated for 48 h; (d) levels of EMT molecules in two ccRCC cell lines treated with fixed dose of Arborinine for 48 h (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001).

Article Snippet: Open reading frame (ORF) clone for KDM1A and UBE2O was obtained from OriGene. shRNAs targeting KDM1A was designed using the Genetic Perturbation Platform (GPP) platform ( https://portals.broadinstitute.org/gpp/public/gene/search ) under the identifiers of TRCN0000046068 and TRCN0000327932.

Techniques: Generated, Western Blot

Journal: Food & Nutrition Research

Article Title: Arborinine from Glycosmis parva leaf extract inhibits clear-cell renal cell carcinoma by inhibiting KDM1A/UBE2O signaling

doi: 10.29219/fnr.v66.8714

Figure Lengend Snippet: Arborinine inhibits KDM1A/UBE2O axis in ccRCC. (a) Reproduced from CistromeDB, shown was transcriptional score of target genes by KDM1A; (b) reproduced from ChIP-Atlas, shown was top 20 transcriptional targets of KDM1A; (c) luciferase activity of candidate target genes in 786O cells in response to KDM1A overexpression; reproduced from TCGA dataset, shown were (d) correlation between expressions of KDM1A and UBE2O and (e) overall survival between cases with higher or lower UBE2O expression; (f) protein level of UBE2O in 786O cells treated with Arborinine; (g) UBE2O mRNA expression in ccRCC cells with silencing of KDM1A and proliferation; replenish of UBE2O by overexpression (OE) in ccRCC cells treated with Arborinine at 20 µM, shown were differences in (h) proliferation, (i) migration, and (j) EMTi profiled by luciferase activity of VIM/CDH1; (k) schematic cartoon of regulatory signaling of Arborinine in ccRCC (* P < 0.05; ** P < 0.01).

Article Snippet: Open reading frame (ORF) clone for KDM1A and UBE2O was obtained from OriGene. shRNAs targeting KDM1A was designed using the Genetic Perturbation Platform (GPP) platform ( https://portals.broadinstitute.org/gpp/public/gene/search ) under the identifiers of TRCN0000046068 and TRCN0000327932.

Techniques: Luciferase, Activity Assay, Over Expression, Expressing, Migration

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: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Inhibitors of histone PTMs

Article Snippet: KDM1A (LSD1) , pCMV6 , Myc-DDK , Mammalian , Origene, #RC208480.

Techniques: Concentration Assay

Journal: Epigenetics & Chromatin

Article Title: The pattern of histone H3 epigenetic posttranslational modifications is regulated by the VRK1 chromatin kinase

doi: 10.1186/s13072-023-00494-7

Figure Lengend Snippet: Plasmids

Article Snippet: KDM1A (LSD1) , pCMV6 , Myc-DDK , Mammalian , Origene, #RC208480.

Techniques: Plasmid Preparation, Expressing

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Workflow of the suppressor screen identifying mutant C . elegans (red) with saliently improved movement. ( B ) Locomotor behavior, measured by thrashing rates in liquid medium, in the C . elegans strains with neuronal expression of human WT SOD1 or ALS-linked mutant SOD1 G85R , in the presence (M1/M1) or absence (+/+) of the suppressor mutation ( n = 16). ( C ) Northern (top panel) and western (middle, bottom panels) blot analyses of total RNA and protein from SOD1 G85R strains, with (M1/M1) or without (+/+) suppressor mutations, demonstrating that the levels of SOD1 G85R mRNA and total protein are not changed by the suppressor mutation (M1/M1). The western blot lanes are from the same gel and exposure. ( D ) Sequence analysis of the M1 strain, revealing that independent mutations in two genes, a lysine-specific demethylase, spr-5 (R646Q) , and a ubiquitin ligase, ufd-2 (W824X) , are required for the full suppressor phenotype. ( E ) C . elegans SPR-5 and UFD-2 and their mammalian homologs lysine-specific demethylase 1 (LSD1) and ubiquitination factor E4 B (UBE4B) share all major protein domains. These include the Swi3-Rsc8-Moira (SWIRM), amine oxidase-like (AOL), and TOWER domains in LSD1/SPR-5 and the Ufd2 Core and U-box domains in UBE4B/UFD-2. Positions of the missense, nonsense, and deletion mutations in mutant C . elegans are indicated. ( F ) Locomotor behavior, measured by thrashing rates, of the C . elegans carrying the SOD1 G85R transgene on the normal background (WT), with the null mutation of either ufd-2(tm1380) or spr-5(by134) , the double mutation ufd-2(tm1380);spr-5(by134) , or the M1 suppressor ufd-2(W824X);spr-5(R646Q ) ( n = 16). ( G ) Western blotting of the supernatant (S) and pellet (P) protein fractions from the C . elegans carrying the SOD1 G85R transgene with the double mutation ufd-2(tm1380);spr-5(by134) compared with controls. While SOD1 G85R protein levels are unchanged in the S fraction, those in the P fraction are decreased in the double suppressor mutant. The P fraction represents only about 1.7% of total SOD1 G85R in the WT sample; therefore, a higher ratio of the P fraction relative to the S fraction (approximately 2:1) is used for the western analysis. ( H ) The overexpression (OE) of WT UFD-2 or SPR-5 in the C . elegans nervous system blocks the protection conferred by the double mutation ufd-2(tm1380);spr-5(by134) ( n = 16). Data represent means ± SEM. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Mutagenesis, Expressing, Northern Blot, Western Blot, Sequencing, Ubiquitin Proteomics, Over Expression

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Top: schematic drawing depicts pan-neuronal expression of YFP in head and ventral neurons in the context of the C . elegans body plan. Left: micrographs show the SOD1 G85R -YFP proteins expressed in WT or the spr-5(by134);ufd-2(tm1380) mutant background. The double-mutant worms show a marked decrease in protein aggregation in neurons. Enlarged sections of head neurons (red framed) and ventral cord neurons (white framed) are shown. Middle: quantification of locomotion in the spr-5(by134);ufd-2(tm1380) and the WT C . elegans with neuronal expression of SOD1 G85R -YFP ( n = 30). Right: a decrease in the protein levels of SOD1 G85R -YFP in the presence of spr-5(by134);ufd-2(tm1380) is shown by western blots of the supernatant (S) and the pellet (P) fractions. ( B and C ) Analyses of the spr-5(by134);ufd-2(tm1380) and the WT C . elegans with neuronal expression of TDP-43-c25-YFP ( n = 30) or PolyQ-YFP ( n = 12) as in (A). ( D ) Neurodegenerative rough-eye phenotype in adults is alleviated by the knockdown of the Drosophila homologs of ufd-2 /UBE4B and spr-5 /LSD1—CG9934 and Su(Var)3-3, respectively, compared to the control (CTRL). Eye-specific expression of TDP-43 M337V , FUS R521C , and RNA interference (RNAi) was driven by GMR-Gal4. ( E ) Drosophila eye pigment quantitation. Data represent means ± SEM. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Expressing, Mutagenesis, Western Blot, Knockdown, Control, Quantitation Assay

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Western blots of cell lysates derived from mock (CTRL), single UBE4B or LSD1, or double UBE4B and LSD1 knockdowns. Supernatant (S) and pellet (P) fractions were probed with indicated antibodies. While the LSD1 or UBE4B single-knockdown reduces the SOD1 G85R aggregates in both supernatant and pellet fractions, the combined knockdown produces the strongest reduction in the aggregates. ( B ) Quantification of SOD1 G85R protein levels by western blotting (A). n = 3 (supernatant); n = 8 (pellet). ( C ) Western blots of a representative cycloheximide chase experiment to determine SOD1 protein half-lives in the double UBE4B and LSD1 knockdown cells versus controls. ( D ) Quantification of SOD1 G85R clearance, as analyzed by western blotting in (C). The graph indicates the relative band intensity of SOD1 G85R at each chase time point. n = 5; Overall p = 0.02 (paired t test, CTRL versus UBE4B and LSD1 double knockdown). Individual p = 0.03 (3 h), p = 0.003 (6 h), p = 0.06 (9 h), and p = 0.004 (12–21 h). ( E ) The half-life of SOD1 G85R is reduced from 8.5 h to 5 h upon knockdown of UBE4B and LSD1. Data represent means ± SEM. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Western Blot, Derivative Assay, Knockdown

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Venn diagram of upstream activators (z-score ≥ 2) that are differentially activated in single—UBE4B or LSD1—knockdowns and double UBE4B and LSD1 knockdowns, compared with the control. Activation state of an upstream regulator is predicted from differential mRNA levels of its downstream target genes. ( B ) The volcano scatter plot indicates fold changes in the levels of gene transcripts affected differentially by the UBE4B and LSD1 double knockdown versus control shRNA. Gray spots represent 22,148 annotated transcripts. Red spots are predicted p53-activated targets, and blue spots are predicted p53-inhibited targets. The enrichment of red spots in the up-regulated genes (right upper quadrant, >1.2-fold change, p ≤ 0.04) and blue spots in the down-regulated genes (left upper quadrant) indicates that the p53-mediated transcription is activated by the UBE4B and LSD1 double knockdown. ( C ) RT-qPCR validation of the expression levels of representative p53 target genes in the UBE4B and LSD1 double-knockdown samples. n = (2 to 6), p ≤ 0.04. ( D ) The p53 protein level is significantly increased by UBE4B and LSD1 double knockdown. Left: representative western blots showing p53 levels, the knockdown of UBE4B and LSD1, and the actin control in HEK293T cells. Right: quantification of the p53 protein levels normalized against actin ( n = 3). ( E ) The p53-mediated transcriptional activity is measured by a luciferase reporter under the control of a p53-response element promoter, which was transfected into HEK293T cells 72–96 h after the initiation of the UBE4B and LSD1 single or double knockdowns ( n = 6). ( F ) Increased interaction of p53 with its activating partner 53BP1 in response to UBE4B and LSD1 knockdown. Left: representative western blots of 53BP1 co-immunoprecipitation. Right: quantification of the 53BP1 co-immunoprecipitation ( n = 2). Data represent means ± SEM. ( G ) RT-qPCR validation of the expression levels of FOXO3a, FOXO4, and PSMD11 ( n = 6). ( H ) The FOXO3a-mediated transcriptional activity was measured with a luciferase reporter under the control of a FOXO-response element promoter. HEK293T cells were transfected with shRNAs for control, LSD1, UBE4B, or both LSD1 and UBE4B, followed by transfection of the luciferase reporter together with a constitutively active form of FOXO3a (TM) 72–96 h later ( n = 6). Data represent means ± SEM. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Control, Activation Assay, Knockdown, shRNA, Quantitative RT-PCR, Biomarker Discovery, Expressing, Western Blot, Activity Assay, Luciferase, Transfection, Immunoprecipitation

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Increased protein levels of proteasome subunits upon the UBE4B and LSD1 double knockdown in HEK293T cells. ( B ) The UBE4B and LSD1 single or double knockdowns increase the proteasomal degradation of a reporter substrate, Suc-LLVY-Luciferin, whose degradation is measured in a luciferase release assay. The double knockdown shows synergistic proteasomal activation when compared with the individual knockdowns ( n = 6). ( C ) The autophagy activity is significantly increased in cells with the UBE4B and LSD1 double knockdown. The in vivo activity of the ATG4B protease, which cleaves LC3 precursors, was quantified via a Gaussia luciferase release assay (see ., and for details). The HCT116 cells were analyzed 48 h after the initiation of the knockdown. ( D ) Quantification of LC3-II levels in HEK293T cells with the UBE4B and LSD1 double knockdown or the mock control. The cells were treated with or without 10 mM 3-methyladenine (3-MA) for 48h before lysis. Western blots indicate an increase in LC3 levels (top panels) in the double UBE4B and LSD1 knockdown cells (middle panels), while the actin control is unchanged (bottom panels). The graph shows the quantification of LC3-II levels as normalized to actin levels ( n = 3). A one-way ANOVA test with matched multiple comparisons and Tukey correction was used for statistics. Data represent means ± SEM. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Knockdown, Luciferase, Release Assay, Activation Assay, Activity Assay, In Vivo, Control, Lysis, Western Blot

Journal: PLoS Biology

Article Title: Regulation of Protein Quality Control by UBE4B and LSD1 through p53-Mediated Transcription

doi: 10.1371/journal.pbio.1002114

Figure Lengend Snippet: ( A ) Left: p53 knockdown reverses the enhanced clearance of SOD1 G85R proteins conferred by the UBE4B and LSD1 double knockdown. Total amounts of shRNAs were adjusted to be equal with nontargeting CTRL shRNAs. Right: quantification of insoluble aggregated SOD1 G85R in pellet fractions from HEK293T cells transfected with control, double (UBE4B/LSD1), or triple (UBE4B/LSD1/p53) shRNAs ( n = 2). ( B ) The degenerative TDP-43 M337V eye phenotype is exacerbated by the knockdown of the Drosophila homolog of p53 (p53 RNAi), or by the overexpression of a dominant-negative p53 mutant (p53.R155H). Expression of p53 RNAi, p53.R155H, and TDP-43 M337V are driven by GMR-Gal4. ( C ) The p53-activating drug Tenovin-1 (TEN1) protects spinal cord motor neurons from SOD1 G85R -induced proteotoxicity. Rat spinal cord cultures were grown as described in Materials and Methods and treated with 0.8 μM TEN1 or vehicle (VEH) DMSO, followed by infection of HSV-SOD1 G85R or the HSV-LacZ control after 24 h. At day 5 post-infection, cells were fixed and stained with a motor neuron-specific anti-neurofilament H (NF-H) antibody. Left: representative images of motor neurons in each condition. Scale bar = 50 μm. Right: quantification of motor neuron survival (one-way ANOVA with multiple comparisons and Tukey correction). Data represent means ± SEM. ( D ) Schematic of the SUNS pathway. A reduction in UBE4B and LSD1 function synergistically activates the p53-mediated transcriptional program, promoting clearance of misfolded and aggregated proteins via increased proteasomal and autophagic clearance. The numerical data used to make this figure can be found in .

Article Snippet: The UBE4B (TF308519) and LSD1 shRNA (TF316984) plasmids and the scrambled control (TR30015) were from Origene.

Techniques: Knockdown, Transfection, Control, Over Expression, Dominant Negative Mutation, Mutagenesis, Expressing, Infection, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: Protein detection of LSD1 in normal and osteoarthritis (OA) cartilage. Tissue sections of human cartilage obtained from non-diseased cartilage ( B ), or OA cartilage from several donors were immunostained for LSD1 ( C – F ). IgG was used as a control where the primary antibody was omitted ( A ) or not ( B ) and using OA cartilage.

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques: Control

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: LSD1 down-regulates the expression of COL9A1 . ( A ) Heatmap of the top 280 regulated genes following LSD1 depletion in articular chondrocytes obtained from three different donors. ( B ) Real-time PCR of a set of collagen encoding genes from at least 3 other independent experiments. Results are presented as relative mRNA levels of COL9A1 , COL2A1 , COL11A2 and AGGRECAN ( ACAN ) normalized to RPLP0 (Student’s t -test * p < 0.05, ** p < 0.01). ( C ) Protein expression of type IX collagen was analyzed by Western blot in cell lysates from human chondrocytes cultured after LSD1 knock-down. Mouse costal chondrocytes (chondro) were used as a positive control ( n = 3). ( D ) Corresponding quantification was performed using ImageJ software (Student’s t -test ** p < 0.01).

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Knockdown, Positive Control, Software

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: LSD1 interacts with SOX9 in human chondrocytes. ( A ) Cytosolic and nuclear fractions were analyzed by Western blot and tested for the expression of SOX9 and LSD1. β-tubulin and Histone H3 were used as cytosolic and nuclear markers, respectively. ( B ) Localization of LSD1 and SOX9 proteins was assessed by immunofluorescence analysis (scale bar: 10µm). Nuclei were counterstained with Hoechst (left panels). ( C ) Immunoprecipitation of SOX9 was realized using an anti-SOX9 antibody, then anti-LSD1 was probed to test if it co-immunoprecipitates ( n = 3). ( D , E ) Knock-down of LSD1 was realized following transfection of human chondrocytes with siRNA. Real-time PCR was performed from at least 5 independent experiments. Results are presented as relative mRNA levels of LSD1 and SOX9 normalized to RPLP0 , after LSD1 knock-down in human chondrocytes (Student’s t -test ** p < 0.01, ns: non significant). Western blot was performed to detect LSD1 or SOX9 protein expression after siRNA transfection. ( F ) Protein synthesis was inhibited with cycloheximide (CHX) incubation and a time course from 0 to 24 h was performed prior to the analysis of SOX9 protein stability by Western blot ( n = 3). ( G ) Corresponding quantification was performed using ImageJ software.

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques: Western Blot, Expressing, Immunofluorescence, Immunoprecipitation, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Incubation, Software

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: LSD1 is recruited onto the COL9A1 promoter region. ( A ) Schematic representation of the COL9A1 promoter which contains four SOX9 binding sites arranged in two pairs. Primers used for PCR are indicated (denoted P1 and -8kb). ( B ) Immunoprecipitation of chromatin was done using anti-SOX9 or anti-LSD1 antibody from human articular chondrocytes. Then, real-time PCR was performed using specific primers P1 of the COL9A1 promoter regions or 8kb upstream ( n = 3, Student’s t -test * p < 0.05). ( C ) Agarose gel shows the corresponding PCR products using the P1 primers.

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques: Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: COL9A1 mRNA levels in LSD1-depleted chondrocytes in response to the proinflammatory cytokine IL-1β. Knock-down of LSD1 was realized following human chondrocyte transfection with siRNA and then treated with IL-1β for 24 h. ( A ) Heatmap of the genes oppositely regulated by LSD1 and IL-1β in articular chondrocytes (NT: non-treated, TT: treated with IL-1β; CT: transfected with siControl, LSD1: transfected with siRNA against LSD1). ( B ) Diagram indicates 57 genes are simultaneously down-regulated by IL-1β and up-regulated following LSD1 depletion. ( C ) Real-time PCR was performed from at least 3 independent experiments of additional donors. Results are presented as relative mRNA levels of COL9A1 normalized to RPLP0 (n = 4, Student’s t -test * p < 0.05 ** p < 0.01). ( D ) Schematic representation showing the down-regulation of COL9A1 by IL-1β is mediated by LSD1.

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques: Knockdown, Transfection, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: The Lysine Specific Demethylase-1 Negatively Regulates the COL9A1 Gene in Human Articular Chondrocytes

doi: 10.3390/ijms21176322

Figure Lengend Snippet: List of primers used for PCR experiments.

Article Snippet: Proteins were then transferred to an Immobilon PVDF membrane (Millipore) and probed with specific primary antibodies against SOX9 (AB5535, Millipore 1/1000), type IX collagen (MAB3304 Millipore, 1/3000), LSD1 (208–13-1-AP, Proteintech, 1/750), Tubulin (T8535, Sigma, 1/2000) or GAPDH (sc-81545, Santa Cruz, 1/2000) overnight at 4 °C after saturation.

Techniques:

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: (A) Neurod6 locus. ChIP-Seq of LSD1 (grey), Setd1a (black), Mef2 (dark blue), H3K27ac (green), H3K4me3 (light blue) is shown. Grey, black and dark blue boxes below Lsd1, Setd1a and Mef2 tracks respectively represent significant peaks that passed all quality checks (see methods).

Article Snippet: LSD1 CDS vector , Origene , Cat# MR210741.

Techniques: ChIP-sequencing

Journal: Neuron

Article Title: Recapitulation and reversal of schizophrenia-related phenotypes in Setd1a -deficient mice

doi: 10.1016/j.neuron.2019.09.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LSD1 CDS vector , Origene , Cat# MR210741.

Techniques: Virus, Recombinant, Luciferase, Plasmid Preparation, Software

Journal: International journal of cancer

Article Title: MiR-137 functions as a tumor suppressor in neuroblastoma by downregulating KDM1A.

doi: 10.1002/ijc.28091

Figure Lengend Snippet: Figure 3. miR-137 directly downregulates KDM1A expression in neuroblastoma cells. (a) Schematic diagram of miR-137 binding site in the KDM1A 30UTR predicted by TargetScan. (pos. 5 position). (b,c) MiR-137 re-expression in neuroblastoma cell lines reduced expression of both KDM1A mRNA measured by RT-qPCR and protein in whole-cell lysates assessed by western blotting. (d) The Pre-miRTM-137 expression plasmid, a luciferase reporter plasmid carrying the KDM1A 30-UTR, and a control plasmid encoding renilla luciferase were cotransfected into HEK-293 or neuroblastoma SHEP cells. Significantly lower luciferase activity (normalized to renilla luciferase activity) was measured com- pared with cells not transfected with any miRNA (“untreated”) or control miRNA transfected cells (student’s t-test: * 5 p < 0.05, ** 5 p < 0.001, *** 5 p < 0.001). For display, normalized luciferase activity of cells not transfected with any miRNA was scaled to 1. (e) RT- qPCR revealed induction of tissue factor pathway inhibitor (TFPI2) upon miR-137 re-expression.

Article Snippet: HEK-293 and SHEP cells were cotransfected with 1 mg/ml of the pMirTarget vector containing the target site of KDM1A 30-UTR (OriGene, Rockville), 0.2 mg/ml pRL-TK vector (Promega, Mannheim, Germany) as an internal control and 20 nM of the Pre-miR using DharmaFECT duo (Thermo Scientific, Schwerte, Germany).

Techniques: Expressing, Binding Assay, Quantitative RT-PCR, Western Blot, Plasmid Preparation, Luciferase, Control, Activity Assay, Transfection

Journal: International journal of cancer

Article Title: MiR-137 functions as a tumor suppressor in neuroblastoma by downregulating KDM1A.

doi: 10.1002/ijc.28091

Figure Lengend Snippet: Figure 4. KDM1A knockdown phenocopies miR-137 re-expression in neuroblastoma cells. (a) Western blot of KDM1A expression in neuro- blastoma whole-cell lysates after either siRNA-mediated KDM1A knockdown (siKDM1A) or scrambled control siRNA (NC). (b) Viability of neu- roblastoma cells upon siRNA-mediated KDM1A knockdown (siKDM1A) compared with scrambled control siRNA transfected cells (NC) determined by MTT assay (custom permutation test). (c) Flow cytometric analysis of neuroblastoma cultures after siRNA-mediated KDM1A knockdown (siKDM1A) or cells transfected with scrambled control siRNA (NC). The subG1 fraction was larger after KDM1A knockdown, indi- cating increase apoptotic cell death. (d) BrdU incorporation assays showed a significant decrease in cell proliferation after KDM1A knock- down in neuroblastoma cells compared with cells transfected with scrambled control siRNA (NC) (e). Cell death ELISA showed that siRNA- mediated KDM1A knockdown induced apoptosis in neuroblastoma cell lines compared with cells transfected with scrambled control siRNA (NC). * 5 p < 0.05; ** 5 p < 0.01; *** 5 p < 0.001

Article Snippet: HEK-293 and SHEP cells were cotransfected with 1 mg/ml of the pMirTarget vector containing the target site of KDM1A 30-UTR (OriGene, Rockville), 0.2 mg/ml pRL-TK vector (Promega, Mannheim, Germany) as an internal control and 20 nM of the Pre-miR using DharmaFECT duo (Thermo Scientific, Schwerte, Germany).

Techniques: Knockdown, Expressing, Western Blot, Control, Transfection, MTT Assay, BrdU Incorporation Assay, Enzyme-linked Immunosorbent Assay

Journal: International journal of cancer

Article Title: MiR-137 functions as a tumor suppressor in neuroblastoma by downregulating KDM1A.

doi: 10.1002/ijc.28091

Figure Lengend Snippet: Figure 5. The presence of miR-137-resistant KDM1A partially rescues the effect of miR-137 expression. (a) Western blot shows KDM1A expression in total protein extracts from SHEP cells stably transfected with a tetracyline-inducible expression plasmid for KDM1A cDNA lack- ing the 30 UTR miR-137 binding site or a tetracyline-inducible expression plasmid for GFP as a control. GAPDH was used as a loading con- trol, and lysates of cells treated with tetracycline (tet) or diluent (EtOH) are shown. (b) MTT assay reflecting conditions of western blot analysis (5A). * 5 p < 0.05; ** 5 p < 0.01; *** 5 p < 0.001.

Article Snippet: HEK-293 and SHEP cells were cotransfected with 1 mg/ml of the pMirTarget vector containing the target site of KDM1A 30-UTR (OriGene, Rockville), 0.2 mg/ml pRL-TK vector (Promega, Mannheim, Germany) as an internal control and 20 nM of the Pre-miR using DharmaFECT duo (Thermo Scientific, Schwerte, Germany).

Techniques: Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Binding Assay, Control, MTT Assay