Review

kdm4a polyclonal antibody (Proteintech)

Name: kdm4a polyclonal antibody
Brand: Proteintech
Rating: 93 (9 reviews)

Proteintech is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Proteintech kdm4a polyclonal antibody

Expression of the <t>KDM4A</t> protein after lentiviral infection. (A) qPCR results of the knockdown of the protein. Expression data are normalized to the Ctrl (b-actin, transcript level). Each experiment was performed in triplicate. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student´s t-test compared to the Ctrl expression level. *p < 0.05. (B) KDM4A protein knockdown during the differentiation protocol. Proteins were extracted at the end of each differentiation stage and detected using specific antibodies against KDM4A. HepG2 and iPSCs WT (wild type) were used as controls. S0-KD, Stage 0 knockdown; S1-KD, Stage 1 knockdown; S2-KD, Stage 2 knockdown; S3-KD, Stage 3 knockdown; S4-KD, Stage 4 knockdown; KDM4A-KD, Histone lysine demethylase 4A knockdown.

Kdm4a Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/kdm4a polyclonal antibody/product/Proteintech
Average 93 stars, based on 9 article reviews

kdm4a polyclonal antibody - by Bioz Stars, 2026-02

93/100 stars

Images

1) Product Images from "Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs"

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

Journal: Frontiers in Endocrinology

doi: 10.3389/fendo.2025.1697097

Expression of the KDM4A protein after lentiviral infection. (A) qPCR results of the knockdown of the protein. Expression data are normalized to the Ctrl (b-actin, transcript level). Each experiment was performed in triplicate. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student´s t-test compared to the Ctrl expression level. *p < 0.05. (B) KDM4A protein knockdown during the differentiation protocol. Proteins were extracted at the end of each differentiation stage and detected using specific antibodies against KDM4A. HepG2 and iPSCs WT (wild type) were used as controls. S0-KD, Stage 0 knockdown; S1-KD, Stage 1 knockdown; S2-KD, Stage 2 knockdown; S3-KD, Stage 3 knockdown; S4-KD, Stage 4 knockdown; KDM4A-KD, Histone lysine demethylase 4A knockdown.

Figure Legend Snippet: Expression of the KDM4A protein after lentiviral infection. (A) qPCR results of the knockdown of the protein. Expression data are normalized to the Ctrl (b-actin, transcript level). Each experiment was performed in triplicate. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student´s t-test compared to the Ctrl expression level. *p < 0.05. (B) KDM4A protein knockdown during the differentiation protocol. Proteins were extracted at the end of each differentiation stage and detected using specific antibodies against KDM4A. HepG2 and iPSCs WT (wild type) were used as controls. S0-KD, Stage 0 knockdown; S1-KD, Stage 1 knockdown; S2-KD, Stage 2 knockdown; S3-KD, Stage 3 knockdown; S4-KD, Stage 4 knockdown; KDM4A-KD, Histone lysine demethylase 4A knockdown.

Techniques Used: Expressing, Infection, Knockdown, Two Tailed Test

Schematic representation of the differentiation process of the iPSCs KDM4A knockdown (iPSCs-KD) to pancreatic β-like cells (PβLCs). iPSCs were cultured in a 12-well plate coated with vitronectin XF™ in a sequential protocol for 13 days. The mTeSR1™ was supplemented with different cofactors and small molecules. Images were captured with a phase contrast microscope (ZEISS AX10). DE, Definitive endoderm; PG, Pancreatic gut tube; PP, Pancreatic progenitor; EP, Endocrine progenitor; PβLC, Pancreatic beta-like cell; d, days. Black bar 50 µm.

Figure Legend Snippet: Schematic representation of the differentiation process of the iPSCs KDM4A knockdown (iPSCs-KD) to pancreatic β-like cells (PβLCs). iPSCs were cultured in a 12-well plate coated with vitronectin XF™ in a sequential protocol for 13 days. The mTeSR1™ was supplemented with different cofactors and small molecules. Images were captured with a phase contrast microscope (ZEISS AX10). DE, Definitive endoderm; PG, Pancreatic gut tube; PP, Pancreatic progenitor; EP, Endocrine progenitor; PβLC, Pancreatic beta-like cell; d, days. Black bar 50 µm.

Techniques Used: Knockdown, Cell Culture, Microscopy

Western blot result of the differentiation process. The figure represents all the proteins detected during different differentiation stages of the iPSCs KDM4A-KD. Proteins are detected individually at each differentiation stage. A HepG2 cell line was used as the control for the experiment. The approximate molecular weight of the protein (kDa) is shown. SOX17–44 kDa; FOXA2–49 kDa; HNF4A 52 kDa; PDX1–40 kDa; SOX9–63 kDa; PTF1A 42 kDa; NKX6.1–50 kDa. S0, Stage 0; S1, Stage 1; S2, Stage 2; S3, Stage 3; S4, Stage 4.

Figure Legend Snippet: Western blot result of the differentiation process. The figure represents all the proteins detected during different differentiation stages of the iPSCs KDM4A-KD. Proteins are detected individually at each differentiation stage. A HepG2 cell line was used as the control for the experiment. The approximate molecular weight of the protein (kDa) is shown. SOX17–44 kDa; FOXA2–49 kDa; HNF4A 52 kDa; PDX1–40 kDa; SOX9–63 kDa; PTF1A 42 kDa; NKX6.1–50 kDa. S0, Stage 0; S1, Stage 1; S2, Stage 2; S3, Stage 3; S4, Stage 4.

Techniques Used: Western Blot, Control, Molecular Weight

Gene expression during differentiation. Line bars represent gene expression in WT iPSCs throughout the differentiation protocol, while dotted bars represent gene expression in KDM4A-KD iPSCs. Expression levels were normalized to the control (β-actin transcript). Each experiment was performed in triplicate. Bars marked with the same symbol (*) indicate a statistically significant difference compared to the control group (two-tailed, unpaired Student’s t-test, *p < 0.05). Bars marked with the same symbol (**) indicate a statistically significant difference between the two groups (WT and KDM4A-KD, two-tailed, unpaired Student’s t-test, **p < 0.05). (A) Stage 0 (DE); (B) Stage 1 Gene expression (PG); (C) Stage 2 Gene expression (PP); (D) Stage 3 Gene expression (EP); (E) Stage 4 Gene expression (PβLC). foxA2, Forkhead box A2; sox17, SRY-box transcription factor 17; hnf4A, Hepatocyte nuclear factor 4 alpha; pdx1, Pancreatic and duodenal homeobox 1; sox9, SRY-box transcription factor 9; nkx6.1, Nk6 homeobox 1 protein; ngn 3, Neurogenin-3; ins, Insulin; ptf1A, Pancreas associated transcription factor 1A; sst, Somatostatin; cgc, Glucagon.

Figure Legend Snippet: Gene expression during differentiation. Line bars represent gene expression in WT iPSCs throughout the differentiation protocol, while dotted bars represent gene expression in KDM4A-KD iPSCs. Expression levels were normalized to the control (β-actin transcript). Each experiment was performed in triplicate. Bars marked with the same symbol (*) indicate a statistically significant difference compared to the control group (two-tailed, unpaired Student’s t-test, *p < 0.05). Bars marked with the same symbol (**) indicate a statistically significant difference between the two groups (WT and KDM4A-KD, two-tailed, unpaired Student’s t-test, **p < 0.05). (A) Stage 0 (DE); (B) Stage 1 Gene expression (PG); (C) Stage 2 Gene expression (PP); (D) Stage 3 Gene expression (EP); (E) Stage 4 Gene expression (PβLC). foxA2, Forkhead box A2; sox17, SRY-box transcription factor 17; hnf4A, Hepatocyte nuclear factor 4 alpha; pdx1, Pancreatic and duodenal homeobox 1; sox9, SRY-box transcription factor 9; nkx6.1, Nk6 homeobox 1 protein; ngn 3, Neurogenin-3; ins, Insulin; ptf1A, Pancreas associated transcription factor 1A; sst, Somatostatin; cgc, Glucagon.

Techniques Used: Gene Expression, Expressing, Control, Two Tailed Test

ELISA measurements of human insulin. Basal insulin was measured one-hour post-treatment with 2 mM glucose (close Barr). Stimulated insulin was measured one-hour post-treatment with 20 mM glucose (open Barr). All measurements were performed by triplicate. PβLC-KD, pancreatic β-like cells KDM4A-KD; PβLC, pancreatic β-like cells; Insulin-secreting cells (INS823/13); iPSC WT, Induced pluripotent cells without differentiation; HEK293T, Human embryonic kidney cells.

Figure Legend Snippet: ELISA measurements of human insulin. Basal insulin was measured one-hour post-treatment with 2 mM glucose (close Barr). Stimulated insulin was measured one-hour post-treatment with 20 mM glucose (open Barr). All measurements were performed by triplicate. PβLC-KD, pancreatic β-like cells KDM4A-KD; PβLC, pancreatic β-like cells; Insulin-secreting cells (INS823/13); iPSC WT, Induced pluripotent cells without differentiation; HEK293T, Human embryonic kidney cells.

Techniques Used: Enzyme-linked Immunosorbent Assay

(A) Immunofluorescence staining showing insulin (green), DAPI (blue), and merge pictures. The results showed that PβLC and PβLC-KD express insulin in response to glucose. However, insulin production in response to glucose by PβLC-KD was lower than that of PβLC, which would be consistent with the results obtained by qRT-PCR. Scale bar = 50 µm. All images were obtained on an Eclipse Ni-E microscope (Nikon) and analyzed with ImageJ software (National Institutes of Health) at 80 ms of exposure. (B) Insulin expression percentage. The fluorescence expressed by insulin was normalized to that expressed by the nucleus. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student’s t-test (p < 0.05*; p < 0.05** n = 3). A. PβLC: Pancreatic β-like cells; B. PβLC-KD: Pancreatic β-like cells KDM4A-KD. C. HepG2: Hepatoblastoma cell line (negative control). D. INS823/13: Rat insulinoma cell line (insulin-secreting cells).

Figure Legend Snippet: (A) Immunofluorescence staining showing insulin (green), DAPI (blue), and merge pictures. The results showed that PβLC and PβLC-KD express insulin in response to glucose. However, insulin production in response to glucose by PβLC-KD was lower than that of PβLC, which would be consistent with the results obtained by qRT-PCR. Scale bar = 50 µm. All images were obtained on an Eclipse Ni-E microscope (Nikon) and analyzed with ImageJ software (National Institutes of Health) at 80 ms of exposure. (B) Insulin expression percentage. The fluorescence expressed by insulin was normalized to that expressed by the nucleus. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student’s t-test (p < 0.05*; p < 0.05** n = 3). A. PβLC: Pancreatic β-like cells; B. PβLC-KD: Pancreatic β-like cells KDM4A-KD. C. HepG2: Hepatoblastoma cell line (negative control). D. INS823/13: Rat insulinoma cell line (insulin-secreting cells).

Techniques Used: Immunofluorescence, Staining, Quantitative RT-PCR, Microscopy, Software, Expressing, Fluorescence, Two Tailed Test, Negative Control

Image Search Results

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Expression of the KDM4A protein after lentiviral infection. (A) qPCR results of the knockdown of the protein. Expression data are normalized to the Ctrl (b-actin, transcript level). Each experiment was performed in triplicate. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student´s t-test compared to the Ctrl expression level. *p < 0.05. (B) KDM4A protein knockdown during the differentiation protocol. Proteins were extracted at the end of each differentiation stage and detected using specific antibodies against KDM4A. HepG2 and iPSCs WT (wild type) were used as controls. S0-KD, Stage 0 knockdown; S1-KD, Stage 1 knockdown; S2-KD, Stage 2 knockdown; S3-KD, Stage 3 knockdown; S4-KD, Stage 4 knockdown; KDM4A-KD, Histone lysine demethylase 4A knockdown.

Article Snippet: Primary antibodies against forkhead box A2 (FOXA2) (710730), SRY-box transcription factor 17 (SOX17) (PA5-72815), hepatocyte nuclear factor alpha (HNF4A) (PA5-82159), SRY-box transcription factor 9 (SOX9) (PA5-81966), pancreas associated transcription factor 1a (PTF1A) (PA5-112677), and Neurogenin 3 (NeuroG3) (703206),c and goat anti-rabbit IgG (H+L) horseradish peroxidase-conjugated secondary antibody ( G21234 ), Luria Broth Base (Miller ́s LB Broth Base) (1295027), PureLinkTM HiPure MaxiPrep kit (K210006), and Fluoromount-GTM, with DAPI (00-4959-52) were obtained from Invitrogen, United States; Recombinant pancreatic and duodenal homeobox 1 (PDX1) (ab219207), and anti-NKX6.1 antibody (ab221549) were obtained from Abcam, United States; CoraLite ® Plus 488-conjugated INS Monoclonal antibody (CL488-66198), and KDM4A polyclonal antibody (29977-1-AP) were acquired from Proteintech, United States; QuickExtractTM RNA extraction kit (QER090150) was obtained from Biosearch Technologies, United States, and OneScript ® Plus Reverse Transcriptase (G237) was obtained from ABN (Canada).

Techniques: Expressing, Infection, Knockdown, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Schematic representation of the differentiation process of the iPSCs KDM4A knockdown (iPSCs-KD) to pancreatic β-like cells (PβLCs). iPSCs were cultured in a 12-well plate coated with vitronectin XF™ in a sequential protocol for 13 days. The mTeSR1™ was supplemented with different cofactors and small molecules. Images were captured with a phase contrast microscope (ZEISS AX10). DE, Definitive endoderm; PG, Pancreatic gut tube; PP, Pancreatic progenitor; EP, Endocrine progenitor; PβLC, Pancreatic beta-like cell; d, days. Black bar 50 µm.

Techniques: Knockdown, Cell Culture, Microscopy

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Western blot result of the differentiation process. The figure represents all the proteins detected during different differentiation stages of the iPSCs KDM4A-KD. Proteins are detected individually at each differentiation stage. A HepG2 cell line was used as the control for the experiment. The approximate molecular weight of the protein (kDa) is shown. SOX17–44 kDa; FOXA2–49 kDa; HNF4A 52 kDa; PDX1–40 kDa; SOX9–63 kDa; PTF1A 42 kDa; NKX6.1–50 kDa. S0, Stage 0; S1, Stage 1; S2, Stage 2; S3, Stage 3; S4, Stage 4.

Techniques: Western Blot, Control, Molecular Weight

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: Gene expression during differentiation. Line bars represent gene expression in WT iPSCs throughout the differentiation protocol, while dotted bars represent gene expression in KDM4A-KD iPSCs. Expression levels were normalized to the control (β-actin transcript). Each experiment was performed in triplicate. Bars marked with the same symbol (*) indicate a statistically significant difference compared to the control group (two-tailed, unpaired Student’s t-test, *p < 0.05). Bars marked with the same symbol (**) indicate a statistically significant difference between the two groups (WT and KDM4A-KD, two-tailed, unpaired Student’s t-test, **p < 0.05). (A) Stage 0 (DE); (B) Stage 1 Gene expression (PG); (C) Stage 2 Gene expression (PP); (D) Stage 3 Gene expression (EP); (E) Stage 4 Gene expression (PβLC). foxA2, Forkhead box A2; sox17, SRY-box transcription factor 17; hnf4A, Hepatocyte nuclear factor 4 alpha; pdx1, Pancreatic and duodenal homeobox 1; sox9, SRY-box transcription factor 9; nkx6.1, Nk6 homeobox 1 protein; ngn 3, Neurogenin-3; ins, Insulin; ptf1A, Pancreas associated transcription factor 1A; sst, Somatostatin; cgc, Glucagon.

Techniques: Gene Expression, Expressing, Control, Two Tailed Test

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: ELISA measurements of human insulin. Basal insulin was measured one-hour post-treatment with 2 mM glucose (close Barr). Stimulated insulin was measured one-hour post-treatment with 20 mM glucose (open Barr). All measurements were performed by triplicate. PβLC-KD, pancreatic β-like cells KDM4A-KD; PβLC, pancreatic β-like cells; Insulin-secreting cells (INS823/13); iPSC WT, Induced pluripotent cells without differentiation; HEK293T, Human embryonic kidney cells.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Endocrinology

Article Title: Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs

doi: 10.3389/fendo.2025.1697097

Figure Lengend Snippet: (A) Immunofluorescence staining showing insulin (green), DAPI (blue), and merge pictures. The results showed that PβLC and PβLC-KD express insulin in response to glucose. However, insulin production in response to glucose by PβLC-KD was lower than that of PβLC, which would be consistent with the results obtained by qRT-PCR. Scale bar = 50 µm. All images were obtained on an Eclipse Ni-E microscope (Nikon) and analyzed with ImageJ software (National Institutes of Health) at 80 ms of exposure. (B) Insulin expression percentage. The fluorescence expressed by insulin was normalized to that expressed by the nucleus. Bars under the same symbol (*) are statistically different under the two-tailed, unpaired Student’s t-test (p < 0.05*; p < 0.05** n = 3). A. PβLC: Pancreatic β-like cells; B. PβLC-KD: Pancreatic β-like cells KDM4A-KD. C. HepG2: Hepatoblastoma cell line (negative control). D. INS823/13: Rat insulinoma cell line (insulin-secreting cells).

Techniques: Immunofluorescence, Staining, Quantitative RT-PCR, Microscopy, Software, Expressing, Fluorescence, Two Tailed Test, Negative Control

ETV2 binds with KDM4A (A and B) Rosa26-CreERT2;Kdm4a/4c f/f mESCs ±4′-OH-tamoxifen were differentiated and analyzed on day 4 for RT-qPCR (A, n = 3) and flow cytometry (B, B′, n = 3). (B) Representative data from three independent experiments. (B′) Quantification data of flow cytometry. Con, wild-type control; DKO, double knockout; FL1, empty channel of flow cytometry. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C) Overexpression of ETV2 increases genes downregulated in Kdm4a/4c DKO mESCs. Kdm4a/4c DKO mESCs overexpressing Etv2 were differentiated and subjected to RT-qPCR. n = 3. ∗∗∗ p < 0.001. (D) Enhanced H3K9me3 in endothelial and hematopoietic genes in the absence of ETV2. Etv2 −/− mESCs were differentiated, harvested at day 4 and cross-linked. Subsequently, the nuclear genomic DNAs were sonicated, and the fragmented genomic DNAs were immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter or enhancer regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05. (E–G) (E) Interaction of ETV2 and KDM4A. HEK/293T cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and western blot analysis. Anti-TUBULIN antibody was used for the internal loading control. (F) In vitro translated FLAG-ETV2 and HA-KDM4A proteins were subjected to immunoprecipitation with an anti-FLAG antibody and western blot analysis with the indicated antibodies. (G) The differentiated wild type mESCs were subjected to immunoprecipitation with an anti-KDM4A antibody, followed by a WB blot analysis with an anti-ETV2 antibody. (H) The cooperative function of ETV2-KDM4A interaction in inducing Flk1 promoter/enhancer ( p/e ) activity. Each construct expressing ETV2 or KDM4A (wt or H188A, a defective mutant of demethylation activity of KDM4A) was introduced into HEK/293T cells together with the Firefly luciferase reporter construct, pGL3-Flk1(p/e) . The Firefly luciferase activity was normalized with Renilla luciferase activity. Data are mean ± SEM of three independent experiments with triplicate assays/experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: ETV2 binds with KDM4A (A and B) Rosa26-CreERT2;Kdm4a/4c f/f mESCs ±4′-OH-tamoxifen were differentiated and analyzed on day 4 for RT-qPCR (A, n = 3) and flow cytometry (B, B′, n = 3). (B) Representative data from three independent experiments. (B′) Quantification data of flow cytometry. Con, wild-type control; DKO, double knockout; FL1, empty channel of flow cytometry. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C) Overexpression of ETV2 increases genes downregulated in Kdm4a/4c DKO mESCs. Kdm4a/4c DKO mESCs overexpressing Etv2 were differentiated and subjected to RT-qPCR. n = 3. ∗∗∗ p < 0.001. (D) Enhanced H3K9me3 in endothelial and hematopoietic genes in the absence of ETV2. Etv2 −/− mESCs were differentiated, harvested at day 4 and cross-linked. Subsequently, the nuclear genomic DNAs were sonicated, and the fragmented genomic DNAs were immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter or enhancer regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05. (E–G) (E) Interaction of ETV2 and KDM4A. HEK/293T cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and western blot analysis. Anti-TUBULIN antibody was used for the internal loading control. (F) In vitro translated FLAG-ETV2 and HA-KDM4A proteins were subjected to immunoprecipitation with an anti-FLAG antibody and western blot analysis with the indicated antibodies. (G) The differentiated wild type mESCs were subjected to immunoprecipitation with an anti-KDM4A antibody, followed by a WB blot analysis with an anti-ETV2 antibody. (H) The cooperative function of ETV2-KDM4A interaction in inducing Flk1 promoter/enhancer ( p/e ) activity. Each construct expressing ETV2 or KDM4A (wt or H188A, a defective mutant of demethylation activity of KDM4A) was introduced into HEK/293T cells together with the Firefly luciferase reporter construct, pGL3-Flk1(p/e) . The Firefly luciferase activity was normalized with Renilla luciferase activity. Data are mean ± SEM of three independent experiments with triplicate assays/experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control, Double Knockout, Over Expression, Sonication, Immunoprecipitation, Amplification, Transfection, Construct, Western Blot, In Vitro, Activity Assay, Expressing, Mutagenesis, Luciferase

The ETV2-mediated generation of FLK1 + cells, hematopoietic and endothelial lineages is regulated by the demethylase activity of KDM4A (A and B) iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated for 4 days were subjected to flow cytometry (A, A′, n = 3) and gene expression analysis (B, n = 3). Dox (1 μg/mL) was treated at day 2. (A) Representative data from three independent experiments is shown. Numbers in the plots denote the percentages of FLK1 + cells. FL1: empty channel. (A′) The quantification data of the flow cytometry. (B) Expression of each gene was normalized against Gapdh , and the fold change of its expression level (+Dox/-Dox) was calculated. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C and D) Flow cytometry analysis for FLK1 and hCD4/SCL (C, C′) and CDH5 (D, D′) in D6 EBs of indicated mESCs. Results are means ± SEM from three independent experiments. FL1: empty channel. ∗ p < 0.05, ∗∗ p < 0.01. (E) Hematopoietic colony assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with ± Dox at D3 and subjected to a hematopoietic replating assay at day 6. Colonies were counted 4 days later. Results are means ± SEM from three independent experiments. ∗∗ p < 0.01. (F) EB sprouting assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with Dox at D3 and subjected to the sprouting assay in a 3D-collagen matrix. The mean sprouting area was measured using ImageJ software 8 days later. 3 EBs/group, n = 3, ∗ p < 0.05.

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: The ETV2-mediated generation of FLK1 + cells, hematopoietic and endothelial lineages is regulated by the demethylase activity of KDM4A (A and B) iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated for 4 days were subjected to flow cytometry (A, A′, n = 3) and gene expression analysis (B, n = 3). Dox (1 μg/mL) was treated at day 2. (A) Representative data from three independent experiments is shown. Numbers in the plots denote the percentages of FLK1 + cells. FL1: empty channel. (A′) The quantification data of the flow cytometry. (B) Expression of each gene was normalized against Gapdh , and the fold change of its expression level (+Dox/-Dox) was calculated. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C and D) Flow cytometry analysis for FLK1 and hCD4/SCL (C, C′) and CDH5 (D, D′) in D6 EBs of indicated mESCs. Results are means ± SEM from three independent experiments. FL1: empty channel. ∗ p < 0.05, ∗∗ p < 0.01. (E) Hematopoietic colony assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with ± Dox at D3 and subjected to a hematopoietic replating assay at day 6. Colonies were counted 4 days later. Results are means ± SEM from three independent experiments. ∗∗ p < 0.01. (F) EB sprouting assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with Dox at D3 and subjected to the sprouting assay in a 3D-collagen matrix. The mean sprouting area was measured using ImageJ software 8 days later. 3 EBs/group, n = 3, ∗ p < 0.05.

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Activity Assay, Flow Cytometry, Gene Expression, Expressing, Hematopoietic Colony Assay, Software

KDM4A regulates H3K9me3 methylation on the target genes of ETV2 iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs were differentiated ± Dox and EBs at day 4 were cross-linked, sonicated, and the fragmented genomic DNAs were subsequently immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05.

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: KDM4A regulates H3K9me3 methylation on the target genes of ETV2 iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs were differentiated ± Dox and EBs at day 4 were cross-linked, sonicated, and the fragmented genomic DNAs were subsequently immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05.

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Methylation, Sonication, Immunoprecipitation, Amplification

ETV2 and KDM4A complex binds to hematopoietic and endothelial genes The genomic DNAs prepared for ChIP-PCR from differentiated iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A ESCs were incubated with rabbit anti-FLAG (for ETV2), rabbit anti-HA (for KDM4A), and rabbit IgG antibody, followed by qPCR with primers corresponding to the promoter or enhancer regions of the indicated genes. The DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05.

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: ETV2 and KDM4A complex binds to hematopoietic and endothelial genes The genomic DNAs prepared for ChIP-PCR from differentiated iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A ESCs were incubated with rabbit anti-FLAG (for ETV2), rabbit anti-HA (for KDM4A), and rabbit IgG antibody, followed by qPCR with primers corresponding to the promoter or enhancer regions of the indicated genes. The DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05.

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Incubation

ETV2 and KDM4A cooperatively promote neovascularization in a mouse model of hindlimb ischemia Mice deficient in endothelial Kdm4a , Etv2 , Etv2 and Kdm4a , wild type were subjected to a mouse model of hindlimb ischemia. (A) Representative images showing physiological status and blood perfusion measured by laser speckle contrast analyzer (LASCA) on day 28 post-injury. n = 5/group. (A′) The blood perfusion ratio of ischemic limbs was measured by LASCA on days 0, 7, 21, and 28 after injury. n = 5/group, ∗∗∗ p < 0.001 (littermate wt control vs. Kdm4a f/f CKO ( Cdh5-Cre;Kdm4a f/f ) and Etv2 f/f CKO ( Cdh5-Cre;Etv2 f/f )), ### p < 0.001 (littermate wt control vs. Kdm4a f/f ;Etv2 f/f CKO ( Cdh5-Cre;Kdm4a f/f ;Etv2 f/f )), $$$ p < 0.001 (Kdm4a f/f ;Etv2 f/f CKO ( Cdh5-Cre;Kdm4a f/f ;Etv2 f/f ) vs. Kdm4a f/f CKO ( Cdh5-Cre;Kdm4a f/f ) and Etv2 f/f CKO ( Cdh5-Cre;Etv2 f/f )). Error bars indicate ± SDs. (B) Representative images of ISOLECTIN B4 + vessels in the ischemic hindlimbs. Adductor muscles from the mice were harvested 28 days after injury and subjected to immunohistochemistry with FITC-conjugated ISOLECTIN B4 to detect vascular structure (green). DAPI (blue) was used for nuclear staining. Scale bars: 100 μm. (B′) Vessel density as represented by the proportion of ISOLECTIN B4 stained area over the total area in the ischemic hindlimbs on day 28 post-injury. ( n = 18 images; 2 images/section × 3 sections/mouse × 3 mice/group). ∗∗∗ p < 0.001. Error bars indicate ± SDs. (C and D) (C) Representative images of the ischemic hindlimb showing muscle necrotic lesions (H&E stain; white arrowhead) and (D) fibrotic lesions stained in blue (Masson’s trichrome stain; black arrowhead). Scale bars: 100 μm. Insets in the photographs in c and d show a magnified image of the area marked with an asterisk.

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: ETV2 and KDM4A cooperatively promote neovascularization in a mouse model of hindlimb ischemia Mice deficient in endothelial Kdm4a , Etv2 , Etv2 and Kdm4a , wild type were subjected to a mouse model of hindlimb ischemia. (A) Representative images showing physiological status and blood perfusion measured by laser speckle contrast analyzer (LASCA) on day 28 post-injury. n = 5/group. (A′) The blood perfusion ratio of ischemic limbs was measured by LASCA on days 0, 7, 21, and 28 after injury. n = 5/group, ∗∗∗ p < 0.001 (littermate wt control vs. Kdm4a f/f CKO ( Cdh5-Cre;Kdm4a f/f ) and Etv2 f/f CKO ( Cdh5-Cre;Etv2 f/f )), ### p < 0.001 (littermate wt control vs. Kdm4a f/f ;Etv2 f/f CKO ( Cdh5-Cre;Kdm4a f/f ;Etv2 f/f )), $$$ p < 0.001 (Kdm4a f/f ;Etv2 f/f CKO ( Cdh5-Cre;Kdm4a f/f ;Etv2 f/f ) vs. Kdm4a f/f CKO ( Cdh5-Cre;Kdm4a f/f ) and Etv2 f/f CKO ( Cdh5-Cre;Etv2 f/f )). Error bars indicate ± SDs. (B) Representative images of ISOLECTIN B4 + vessels in the ischemic hindlimbs. Adductor muscles from the mice were harvested 28 days after injury and subjected to immunohistochemistry with FITC-conjugated ISOLECTIN B4 to detect vascular structure (green). DAPI (blue) was used for nuclear staining. Scale bars: 100 μm. (B′) Vessel density as represented by the proportion of ISOLECTIN B4 stained area over the total area in the ischemic hindlimbs on day 28 post-injury. ( n = 18 images; 2 images/section × 3 sections/mouse × 3 mice/group). ∗∗∗ p < 0.001. Error bars indicate ± SDs. (C and D) (C) Representative images of the ischemic hindlimb showing muscle necrotic lesions (H&E stain; white arrowhead) and (D) fibrotic lesions stained in blue (Masson’s trichrome stain; black arrowhead). Scale bars: 100 μm. Insets in the photographs in c and d show a magnified image of the area marked with an asterisk.

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Control, Muscles, Immunohistochemistry, Staining

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-human KDM4A antibody , Bethyl Laboratories , Cat# A300-861A; RRID: N/A.

Techniques: Plasmid Preparation, Recombinant, Virus, Control, Transfection, Staining, Knock-Out, Protease Inhibitor, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Cell Culture, DNA Purification, DNA Methylation Assay, Reporter Assay, Software

SUMO modification of KDM4A-regulated host genes differentially expressed in KSHV lytic reactivated BCBL-1 cells. (A) Summary of RNA-seq data. TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R cells were treated with 0.2 μg/mL Dox for 24 h. Cells cultured without any treatment were used as control (Ctrl). Total RNA was extracted and used for RNA-seq on the Illumina HiSeq2000 platform 24 h after treatment. Total reads of each sample were showed in the top boxes. Paired-end reads were aligned to the human reference genome (hg38) using CLC Genomics Workbench 11 (Qiagen) and annotated with RefSeq82 using Partek Genomics Suite 7 (Partek). RPKM greater than 0.1 in any one of the samples was considered as expressed and used for subsequent analysis. Pie chart shows mRNA expression data. The numbers (percentages) of mRNAs that were up- or downregulated more than 2-fold are shown. (B) Heatmap of hierarchical cluster analysis of the mRNA expression data from the treatment conditions described in .

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: SUMO modification of KDM4A-regulated host genes differentially expressed in KSHV lytic reactivated BCBL-1 cells. (A) Summary of RNA-seq data. TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R cells were treated with 0.2 μg/mL Dox for 24 h. Cells cultured without any treatment were used as control (Ctrl). Total RNA was extracted and used for RNA-seq on the Illumina HiSeq2000 platform 24 h after treatment. Total reads of each sample were showed in the top boxes. Paired-end reads were aligned to the human reference genome (hg38) using CLC Genomics Workbench 11 (Qiagen) and annotated with RefSeq82 using Partek Genomics Suite 7 (Partek). RPKM greater than 0.1 in any one of the samples was considered as expressed and used for subsequent analysis. Pie chart shows mRNA expression data. The numbers (percentages) of mRNAs that were up- or downregulated more than 2-fold are shown. (B) Heatmap of hierarchical cluster analysis of the mRNA expression data from the treatment conditions described in .

Article Snippet: Chromatin DNA prepared from 1 × 10 7 TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R cells was used for ChIP assays using anti-KDM4A rabbit polyclonal antibody ( ) and rabbit nonimmune serum IgG (Alpha Diagnostic International).

Techniques: Modification, RNA Sequencing, Cell Culture, Control, Expressing

Pathway enrichment of differentially expressed genes (DEGs) between KDM4A-WT and -K471R BCBL-1 after KSHV reactivation. The numbers on the x axis indicate -log 10 P -value.

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: Pathway enrichment of differentially expressed genes (DEGs) between KDM4A-WT and -K471R BCBL-1 after KSHV reactivation. The numbers on the x axis indicate -log 10 P -value.

Techniques:

SUMOylation of KDM4A enhanced BCBL-1 cell movement and angiogenesis of HMEC-1 cells. (A) Trajectory tracking of TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox cultivated on top of collagen. The amoeboid cell movement was recorded via a brightfield microscopy for 24 h at 5-min intervals. All the movement cells from 6 microscopic fields were shown. (B) The quantitative data of the directional migration distance was calculated with Image J software. (C) Representative images (×100 magnification) of in vitro tube formation assay in HMEC-1 cells treated with conditioned medium (CM) collected from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox (0.2 μg/mL) for 72 h (left panel). Quantification of tube length (right panel). (D) Generation of KDM4A knockout TREx-MH-K-Rta BCBL-1 cell line by CRISPR/Cas9 system. Immunoblotting of KDM4A in different TREx-MH-K-Rta BCBL-1 knockout clones. (E) The knockout clone (#6) was confirmed by Sanger sequencing of PCR amplicons. (F) Immunoblotting of KDM4A expression in the control (Ctrl), Flag-tag-expressing vector (Flag), KDM4A-WT, and KDM4A-K471R transduced TREx-MH-K-Rta-KDM4A KO BCBL-1 cells with or without Dox (0.2 μg/mL) treatment for 72 h. (G) Representative images (×100 magnification) of in vitro tube formation assay in HMEC-1 cells treated with CM collected from TREx-MH-K-Rta-KDM4A ko -Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox (0.2 μg/mL) for 72 h (left panel). Quantification of tube length (right panel).

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: SUMOylation of KDM4A enhanced BCBL-1 cell movement and angiogenesis of HMEC-1 cells. (A) Trajectory tracking of TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox cultivated on top of collagen. The amoeboid cell movement was recorded via a brightfield microscopy for 24 h at 5-min intervals. All the movement cells from 6 microscopic fields were shown. (B) The quantitative data of the directional migration distance was calculated with Image J software. (C) Representative images (×100 magnification) of in vitro tube formation assay in HMEC-1 cells treated with conditioned medium (CM) collected from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox (0.2 μg/mL) for 72 h (left panel). Quantification of tube length (right panel). (D) Generation of KDM4A knockout TREx-MH-K-Rta BCBL-1 cell line by CRISPR/Cas9 system. Immunoblotting of KDM4A in different TREx-MH-K-Rta BCBL-1 knockout clones. (E) The knockout clone (#6) was confirmed by Sanger sequencing of PCR amplicons. (F) Immunoblotting of KDM4A expression in the control (Ctrl), Flag-tag-expressing vector (Flag), KDM4A-WT, and KDM4A-K471R transduced TREx-MH-K-Rta-KDM4A KO BCBL-1 cells with or without Dox (0.2 μg/mL) treatment for 72 h. (G) Representative images (×100 magnification) of in vitro tube formation assay in HMEC-1 cells treated with CM collected from TREx-MH-K-Rta-KDM4A ko -Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells treated with or without Dox (0.2 μg/mL) for 72 h (left panel). Quantification of tube length (right panel).

Techniques: Microscopy, Migration, Software, In Vitro, Tube Formation Assay, Knock-Out, CRISPR, Western Blot, Clone Assay, Sequencing, Expressing, Control, FLAG-tag, Plasmid Preparation

Identification of IL-10 as a novel target gene of KDM4A potentially involved in angiogenesis of endothelial HMEC-1 cells. (A) Real-time reverse transcriptase-quantitative PCR (RT-qPCR) analysis of the expression of angiogenesis-related genes in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and KDM4A-K471R cells treated with Dox (0.2 μg/mL) for 0 and 24 h. The results are expressed as fold change (FC) compared to KDM4A-WT cells without Dox (assigned a value of 1). (B) The expression of IL-10 in the TREx-MH-K-Rta (WT) and TREx-MH-K-Rta-shKDM4A BCBL-1 cells was analyzed by RT-qPCR (left panel). Culture supernatants were collected, and levels of IL-10 were measured by ELISA (right panel). The results are expressed as FC compared to wild-type (WT) cells (assigned a value of 1). (C) Histograms of ChIP-seq profiles for KDM4A binding at IL-10 loci in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells. (D and E) ChIP assay was performed with chromatin prepared from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells (D) and from TREx-MH-K-Rta BCBL-1 cells (E) using rabbit IgG and anti-KDM4A antibody. ChIP DNA was quantified by RT-qPCR using primer pairs specific for promoter regions of IL-10. (F) ChIP-qPCR assay was performed on noninduced and Dox-induced TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells using rabbit IgG and anti-H3K9me3 antibody. ChIP DNA levels were determined as described in (D) and (E). (G) Culture supernatants from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and KDM4A-K471R cells with or without Dox (0.2 μg/mL) treatment for 72 h were collected and the levels of IL-10 were measured by ELISA. (H) TREx-MH-K-Rta BCBL-1 cells were transient transduced with lentivirus overexpressing shIL-10 (clone #1 and #2). Culture supernatants from the KSHV-reactivated control (Ctrl) and IL-10 knockdown (shIL-10) BCBL-1 cells were collected, and IL-10 levels were determined by ELISA. (I) Quantification of the tube length of HMEC-1 treated with CM collected from cells treated as described in (H). (J) IL-10 levels in KSHV-reactivated BCBL-1 cells with or without preincubation with 0.05, 0.15, and 0.2 μg/mL IL-10 nAb for 10 min at 25°C were determined by ELISA. (K) Quantification of the tube length of HMEC-1 treated with CM collected from TREx-MH-K-Rta BCBL-1 cells with or without preincubation with 0.2 μg/mL IL-10 nAb for 10 min at 25°C.

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: Identification of IL-10 as a novel target gene of KDM4A potentially involved in angiogenesis of endothelial HMEC-1 cells. (A) Real-time reverse transcriptase-quantitative PCR (RT-qPCR) analysis of the expression of angiogenesis-related genes in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and KDM4A-K471R cells treated with Dox (0.2 μg/mL) for 0 and 24 h. The results are expressed as fold change (FC) compared to KDM4A-WT cells without Dox (assigned a value of 1). (B) The expression of IL-10 in the TREx-MH-K-Rta (WT) and TREx-MH-K-Rta-shKDM4A BCBL-1 cells was analyzed by RT-qPCR (left panel). Culture supernatants were collected, and levels of IL-10 were measured by ELISA (right panel). The results are expressed as FC compared to wild-type (WT) cells (assigned a value of 1). (C) Histograms of ChIP-seq profiles for KDM4A binding at IL-10 loci in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells. (D and E) ChIP assay was performed with chromatin prepared from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells (D) and from TREx-MH-K-Rta BCBL-1 cells (E) using rabbit IgG and anti-KDM4A antibody. ChIP DNA was quantified by RT-qPCR using primer pairs specific for promoter regions of IL-10. (F) ChIP-qPCR assay was performed on noninduced and Dox-induced TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells using rabbit IgG and anti-H3K9me3 antibody. ChIP DNA levels were determined as described in (D) and (E). (G) Culture supernatants from TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and KDM4A-K471R cells with or without Dox (0.2 μg/mL) treatment for 72 h were collected and the levels of IL-10 were measured by ELISA. (H) TREx-MH-K-Rta BCBL-1 cells were transient transduced with lentivirus overexpressing shIL-10 (clone #1 and #2). Culture supernatants from the KSHV-reactivated control (Ctrl) and IL-10 knockdown (shIL-10) BCBL-1 cells were collected, and IL-10 levels were determined by ELISA. (I) Quantification of the tube length of HMEC-1 treated with CM collected from cells treated as described in (H). (J) IL-10 levels in KSHV-reactivated BCBL-1 cells with or without preincubation with 0.05, 0.15, and 0.2 μg/mL IL-10 nAb for 10 min at 25°C were determined by ELISA. (K) Quantification of the tube length of HMEC-1 treated with CM collected from TREx-MH-K-Rta BCBL-1 cells with or without preincubation with 0.2 μg/mL IL-10 nAb for 10 min at 25°C.

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, ChIP-sequencing, Binding Assay, ChIP-qPCR, Transduction, Control, Knockdown

Dissection of KSHV lytic reactivation stages in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cell lines treated with Dox (0.2 μg/mL) for 24 h. (A) The number (cell #) and percentage (%) of TREx-MH-K-Rta-shKDM4A-KDM4A-WT and -KDM4A-K471R BCBL-1 cells in single-cell RNA sequencing (scRNA-seq). (B) T-distributed stochastic neighbor embedding (t-SNE) plot demonstrating cells in different lytic stages (colored and labeled by their marker genes). (C) The heatmap shows the expression (standardized average count) of KSHV lytic genes in each cluster. Row: KSHV genes. Column: cells.

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: Dissection of KSHV lytic reactivation stages in TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cell lines treated with Dox (0.2 μg/mL) for 24 h. (A) The number (cell #) and percentage (%) of TREx-MH-K-Rta-shKDM4A-KDM4A-WT and -KDM4A-K471R BCBL-1 cells in single-cell RNA sequencing (scRNA-seq). (B) T-distributed stochastic neighbor embedding (t-SNE) plot demonstrating cells in different lytic stages (colored and labeled by their marker genes). (C) The heatmap shows the expression (standardized average count) of KSHV lytic genes in each cluster. Row: KSHV genes. Column: cells.

Techniques: Dissection, RNA Sequencing, Labeling, Marker, Expressing

Trajectory analysis of TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells. The trajectory plot of combined (left panel), TREx-MH-K-Rta-shKDM4A-KDM4A-WT (middle panel), and -KDM4A-K471R (right panel) BCBL-1 cells. (A) The trajectory plots are colored with purple, red, and orange, indicating states 1, 2, and 3, respectively. (B) The trajectory plots are colored with blue, green, and yellow, indicating immediate early (IE), early (E), and late (L), respectively. (C) The trajectory plots are colored with the expression of KSHV genes.

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: Trajectory analysis of TREx-MH-K-Rta-shKDM4A-Flag-KDM4A-WT and -KDM4A-K471R BCBL-1 cells. The trajectory plot of combined (left panel), TREx-MH-K-Rta-shKDM4A-KDM4A-WT (middle panel), and -KDM4A-K471R (right panel) BCBL-1 cells. (A) The trajectory plots are colored with purple, red, and orange, indicating states 1, 2, and 3, respectively. (B) The trajectory plots are colored with blue, green, and yellow, indicating immediate early (IE), early (E), and late (L), respectively. (C) The trajectory plots are colored with the expression of KSHV genes.

Techniques: Expressing

KDM4A is required for the survival and virus production of BCBL-1 cells after KSHV reactivation. (A) Proliferation of TREx-MH-K-Rta and TREx-MH-K-Rta KDM4A knockout (TREx-MH-K-Rta-KDM4A KO ) BCBL-1 cell lines before after Dox treatment for 72 h were assessed by MTT assay. (B) Viable cells treated as described in (A) were counted by trypan blue dye exclusion assay using Countess 3 FL. (C) TREx-MH-K-Rta and TREx-MH-K-Rta-KDM4A KO cells were treated with Dox (0.2 μg/mL) for 0 and 72 h, followed by the stain of annexin V and propidium iodide using an apoptotic detection kit. The fluorescence intensity of the stained cells was analyzed using fluorescence-activated cell sorting (FACS). Cells without staining were used as negative control. (D) Supernatants from TREx-MH-K-Rta and KDM4A KO BCBL-1 cells treated as described in (A) for 72 h were collected, filtered, and the viral titers were determined by analyzing the virion-associated DNA levels using TaqMan qPCR. (E) TREx-MH-K-Rta BCBL-1 cells were treated with 0.2 μg/mL Dox, 60 μM NCDM-32B, or both for 0 and 72 h. Cell numbers were counted by trypan blue dye exclusion assay using Countess 3 FL. (F) TREx-MH-K-Rta BCBL-1 cells were treated with 500 ng/mL TSA, 1 mM NaB, 60 μM NCDM-32B/500 ng/mL TSA or 60 μM NCDM-32B/1 mM NaB. Cell numbers were counted as described in (E). Cells without treatment used as a control (Ctrl). (G) Supernatants from TREx-MH-K-Rta BCBL-1 cells treated as described in (F) were collected and filtered, and the viral titers were determined as described in (D). (Data represent mean±SEM; n = 3; *, P < 0.05; ***, P < 0.005).

Journal: Journal of Virology

Article Title: SUMO Modification of Histone Demethylase KDM4A in Kaposi’s Sarcoma-Associated Herpesvirus-Induced Primary Effusion Lymphoma

doi: 10.1128/jvi.00755-22

Figure Lengend Snippet: KDM4A is required for the survival and virus production of BCBL-1 cells after KSHV reactivation. (A) Proliferation of TREx-MH-K-Rta and TREx-MH-K-Rta KDM4A knockout (TREx-MH-K-Rta-KDM4A KO ) BCBL-1 cell lines before after Dox treatment for 72 h were assessed by MTT assay. (B) Viable cells treated as described in (A) were counted by trypan blue dye exclusion assay using Countess 3 FL. (C) TREx-MH-K-Rta and TREx-MH-K-Rta-KDM4A KO cells were treated with Dox (0.2 μg/mL) for 0 and 72 h, followed by the stain of annexin V and propidium iodide using an apoptotic detection kit. The fluorescence intensity of the stained cells was analyzed using fluorescence-activated cell sorting (FACS). Cells without staining were used as negative control. (D) Supernatants from TREx-MH-K-Rta and KDM4A KO BCBL-1 cells treated as described in (A) for 72 h were collected, filtered, and the viral titers were determined by analyzing the virion-associated DNA levels using TaqMan qPCR. (E) TREx-MH-K-Rta BCBL-1 cells were treated with 0.2 μg/mL Dox, 60 μM NCDM-32B, or both for 0 and 72 h. Cell numbers were counted by trypan blue dye exclusion assay using Countess 3 FL. (F) TREx-MH-K-Rta BCBL-1 cells were treated with 500 ng/mL TSA, 1 mM NaB, 60 μM NCDM-32B/500 ng/mL TSA or 60 μM NCDM-32B/1 mM NaB. Cell numbers were counted as described in (E). Cells without treatment used as a control (Ctrl). (G) Supernatants from TREx-MH-K-Rta BCBL-1 cells treated as described in (F) were collected and filtered, and the viral titers were determined as described in (D). (Data represent mean±SEM; n = 3; *, P < 0.05; ***, P < 0.005).

Techniques: Virus, Knock-Out, MTT Assay, Exclusion Assay, Staining, Fluorescence, FACS, Negative Control, Control

Baseline characteristics of the patients with OSCC and distribution of KDM4A by selected study variables

Journal: Oncotarget

Article Title: KDM4A as a prognostic marker of oral squamous cell carcinoma: Evidence from tissue microarray studies in a multicenter cohort

doi: 10.18632/oncotarget.18302

Figure Lengend Snippet: Baseline characteristics of the patients with OSCC and distribution of KDM4A by selected study variables

Article Snippet: Tissue sections were incubated with the primary antibody rabbit polyclonal KDM4A (Bethyl, Montgomery, TX, USA), diluted in 50mM Tris-HCl (1:50, pH 7.6), 150mM NaCl, and 0.1% Tween20 at 4°C overnight, followed by incubations with the ChemMate EnVision/HRP, Rabbit/Mouse (ENV) reagent of Envision Detection Kit (Dako Corporation, Carpinteria, CA, USA) for 60 minutes.

Techniques: Expressing, Cell Differentiation

Univariate analysis of selected characteristics and overall survival of patients with OSCC

Journal: Oncotarget

Article Title: KDM4A as a prognostic marker of oral squamous cell carcinoma: Evidence from tissue microarray studies in a multicenter cohort

doi: 10.18632/oncotarget.18302

Figure Lengend Snippet: Univariate analysis of selected characteristics and overall survival of patients with OSCC

Techniques: Cell Differentiation, Expressing

Kaplan-Meier estimates of disease-free survival (DFS) in relation to KDM4A expression

Journal: Oncotarget

Article Title: KDM4A as a prognostic marker of oral squamous cell carcinoma: Evidence from tissue microarray studies in a multicenter cohort

doi: 10.18632/oncotarget.18302

Figure Lengend Snippet: Kaplan-Meier estimates of disease-free survival (DFS) in relation to KDM4A expression

Techniques: Expressing

Multivariate analysis of overall survival of patients with OSCC

Journal: Oncotarget

Article Title: KDM4A as a prognostic marker of oral squamous cell carcinoma: Evidence from tissue microarray studies in a multicenter cohort

doi: 10.18632/oncotarget.18302

Figure Lengend Snippet: Multivariate analysis of overall survival of patients with OSCC

Techniques: Cell Differentiation, Expressing

ROC curves for models with and without KDM4A expression in the cohorts

Journal: Oncotarget

Article Title: KDM4A as a prognostic marker of oral squamous cell carcinoma: Evidence from tissue microarray studies in a multicenter cohort

doi: 10.18632/oncotarget.18302

Figure Lengend Snippet: ROC curves for models with and without KDM4A expression in the cohorts

Techniques: Expressing

( a ) Actively growing U2OS cells (confluency 60–70%) were fixed following extraction with 1% Triton X-100 and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panel 1) and human Pol-I largest subunit A194 (panel 2); nuclear DNA was stained by DAPI (panel 3). Images were acquired with a Leica fluorescent microscope (DM5000- 20 × objective). Representative nuclei are shown with associated merged images (panel 4). Scale bar, 5 μM. ( b ) A diagram of the human rDNA repeat is shown indicating the positions of eight sets of specific PCR primer/probes used for qPCR analysis of immunoprecipitated DNA. 5′ETS, 5′-external transcribed spacer; IGS, intergenic spacer; Prom, the rRNA promoter, term, the terminator. Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. ( c ) ChIP assays were performed using antibodies specific to human KDM4A from two different sources and analysed by qPCR using eight sets of specific probes and primers derived from different regions of rDNA repeats (see the diagram above). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. The s.d.'s from three independent experiments are shown; n =3. ( d , e ) KDM4A was immunoprecipitated from nuclear extract of U2OS cells using KMD4A-specific antibodies (Bethyl). Immunoprecipitated complexes were analysed by western blotting using either antibodies specific to human Pol-I largest subunit A194 and UBF (lane 4), or to SL1 subunits TAF 1 110 and TAF 1 63 (lane 7). Input—lanes 1, 2 and 6, negative control (anti-HA antibodies)—lanes 3 and 8. Purified SL1—lane 5. Positions of prestained molecular weight markers (PageRuler Plus, Fermentas) are indicated.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Actively growing U2OS cells (confluency 60–70%) were fixed following extraction with 1% Triton X-100 and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panel 1) and human Pol-I largest subunit A194 (panel 2); nuclear DNA was stained by DAPI (panel 3). Images were acquired with a Leica fluorescent microscope (DM5000- 20 × objective). Representative nuclei are shown with associated merged images (panel 4). Scale bar, 5 μM. ( b ) A diagram of the human rDNA repeat is shown indicating the positions of eight sets of specific PCR primer/probes used for qPCR analysis of immunoprecipitated DNA. 5′ETS, 5′-external transcribed spacer; IGS, intergenic spacer; Prom, the rRNA promoter, term, the terminator. Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. ( c ) ChIP assays were performed using antibodies specific to human KDM4A from two different sources and analysed by qPCR using eight sets of specific probes and primers derived from different regions of rDNA repeats (see the diagram above). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Signal representing the transcribed region (TrR) is the average of the combined signal from 5′ETS, 18S, 5.8S and 28S rRNA. Signal representing the non-transcribed region (nTrR) is the average of the combined signal from IGS1 and IGS2. The s.d.'s from three independent experiments are shown; n =3. ( d , e ) KDM4A was immunoprecipitated from nuclear extract of U2OS cells using KMD4A-specific antibodies (Bethyl). Immunoprecipitated complexes were analysed by western blotting using either antibodies specific to human Pol-I largest subunit A194 and UBF (lane 4), or to SL1 subunits TAF 1 110 and TAF 1 63 (lane 7). Input—lanes 1, 2 and 6, negative control (anti-HA antibodies)—lanes 3 and 8. Purified SL1—lane 5. Positions of prestained molecular weight markers (PageRuler Plus, Fermentas) are indicated.

Article Snippet: Cells were then incubated at 4 °C overnight with the indicated primary antibodies: Rabbit polyclonal anti-JMJD2A (Bethyl A-300-861-A; 0.5 μg ml −1 ), Rabbit polyclonal anti-KDM4A antibodies (Sigma, HPA007610, 0.5 μg ml −1 ), Rabbit polyclonal anti-HA (Cell Signaling C29F4, 1 μg ml −1 ), RPA 194 Santa Cruz SC-48385 1 μg ml −1 , mouse monoclonal anti-UBF (SC-13125 1 μg ml −1 ), mouse monoclonal anti-BrdU 2 μg ml −1 (BU-33, Sigma), mouse monoclonal anti-myc antibodies (9E10, Santa Cruz).

Techniques: Immunofluorescence, Staining, Microscopy, Immunoprecipitation, Derivative Assay, Negative Control, Western Blot, Purification, Molecular Weight

( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on ongoing rRNA synthesis. RNA was extracted 2 and 4 h after 3 H-uridine addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots as in . ( b ) The relative efficiencies of rRNA synthesis were quantitated as in and transcript levels are indicated for 47S/45S pre-rRNA. The data, expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( c ) The relative efficiencies of cell growth were determined using MTT assay. The s.d.'s for four independent samples are shown; n =4. ( d ) Growing U2OS cells (∼70% confluent) were electroporated with an expression vector encoding either wild-type KDM4A (WT) or catalytically inactive mutant (MUT). Cells were grown for 48 h and then 5-FUrd was added for 30 min and cells were fixed and analysed by indirect immunofluorescence using antibodies specific to 5-FUrd (anti-BrdU antibodies); nuclear DNA was stained by DAPI. ( e ) Box-and-whiskers plot of the quantification of 5-FUrd staining per cell ( n =100–200) obtained from the experiment shown in d using R open source software (a.u. stands for arbitrary units). Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are not significantly different ( P value=0.637, Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on ongoing rRNA synthesis. RNA was extracted 2 and 4 h after 3 H-uridine addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots as in . ( b ) The relative efficiencies of rRNA synthesis were quantitated as in and transcript levels are indicated for 47S/45S pre-rRNA. The data, expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( c ) The relative efficiencies of cell growth were determined using MTT assay. The s.d.'s for four independent samples are shown; n =4. ( d ) Growing U2OS cells (∼70% confluent) were electroporated with an expression vector encoding either wild-type KDM4A (WT) or catalytically inactive mutant (MUT). Cells were grown for 48 h and then 5-FUrd was added for 30 min and cells were fixed and analysed by indirect immunofluorescence using antibodies specific to 5-FUrd (anti-BrdU antibodies); nuclear DNA was stained by DAPI. ( e ) Box-and-whiskers plot of the quantification of 5-FUrd staining per cell ( n =100–200) obtained from the experiment shown in d using R open source software (a.u. stands for arbitrary units). Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are not significantly different ( P value=0.637, Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Techniques: Imaging, MTT Assay, Expressing, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining, Software

( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on activation of rRNA synthesis. U2OS cells were transfected either with non-targeting (siScr) or KDM4A-specific siRNA3 (siA3). 24 h post-transfection cells were starved. Transcription was activated by addition of serum and 3 H-uridine at time point 0. ( b ) RNA was extracted 15, 30, 60 and 90 min after serum addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots (top panel) using X-ray film. Total 18S and 28S rRNAs were detected by ethidium bromide staining (bottom panel). ( c ) To determine the relative efficiencies of rRNA synthesis, RNA blots were imaged using tritium image plate (Fuji) and quantitated with aid of phosphoimager (Fuji) and Aida software (Raytec). Transcript levels are indicated for 47S/45S pre-rRNA. The data are expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( d ). Schematic representation of the labelling of cells with 5-FUrd to determine the effect of KDM4A depletion on activation of rRNA synthesis in individual cells. U2OS cells were serum-starved for 16 h, electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against KDM4A (siA1 and siA2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd for 30 min. ( e ). Cells were stained for 5-FUrd incorporation using anti-BrdU antibodies. Nuclear DNA was stained with DAPI. Representative immunofluorescence images are shown. Scale bar, 5 μM. ( f ). Quantification of 5-FUrd staining shown in e , done as in ; P value (Wilcoxon test)= * 4.84 × 10 −11 , ** 6.12 × 10 −12 . Note the presence of many outliers in samples transfected by KDM4A siRNA, which most likely correspond to untransfected cells. The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Schematic representation of the labelling of cells with 3 H-uridine to determine the effect of KDM4A depletion on activation of rRNA synthesis. U2OS cells were transfected either with non-targeting (siScr) or KDM4A-specific siRNA3 (siA3). 24 h post-transfection cells were starved. Transcription was activated by addition of serum and 3 H-uridine at time point 0. ( b ) RNA was extracted 15, 30, 60 and 90 min after serum addition and de nov o rRNA transcripts were detected by tritium imaging of RNA blots (top panel) using X-ray film. Total 18S and 28S rRNAs were detected by ethidium bromide staining (bottom panel). ( c ) To determine the relative efficiencies of rRNA synthesis, RNA blots were imaged using tritium image plate (Fuji) and quantitated with aid of phosphoimager (Fuji) and Aida software (Raytec). Transcript levels are indicated for 47S/45S pre-rRNA. The data are expressed as a percentage of the highest value (set at 100%). The s.d.'s for three independent experiments are shown; n =3. ( d ). Schematic representation of the labelling of cells with 5-FUrd to determine the effect of KDM4A depletion on activation of rRNA synthesis in individual cells. U2OS cells were serum-starved for 16 h, electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against KDM4A (siA1 and siA2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd for 30 min. ( e ). Cells were stained for 5-FUrd incorporation using anti-BrdU antibodies. Nuclear DNA was stained with DAPI. Representative immunofluorescence images are shown. Scale bar, 5 μM. ( f ). Quantification of 5-FUrd staining shown in e , done as in ; P value (Wilcoxon test)= * 4.84 × 10 −11 , ** 6.12 × 10 −12 . Note the presence of many outliers in samples transfected by KDM4A siRNA, which most likely correspond to untransfected cells. The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Techniques: Activation Assay, Transfection, Imaging, Staining, Software, Immunofluorescence

( a ). U2OS cells were serum starved for 24 h ( t =0) and refed with serum. Cells were fixed at 30 and 60 min following serum addition, and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panels 1) and human Pol-I largest subunit A194 (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panel 4). Scale bar, 5 μM. ( b ) ChIP assays were performed using antibodies specific to human KDM4A (Ab1, Bethyl Lab) and chromatin isolated from starved and starved–refed cells (30 and 60 min after serum addition) and analysed as in to determine KDM4A binding to rDNA promoter (Pr), transcribed region (TrR) or non-transcribed region (nTrR). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( c ) ChIP assays were performed using antibodies specific to human KDM4A as in b and chromatin immunoprecipitated from starved and starved–refed cells (60 min after serum addition) were analysed by qPCR for the presence either CDC6 or P0 or GAPDH promoters representing a positive and negative controls of the anti-KDM4A ChIP, respectively. The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ). U2OS cells were serum starved for 24 h ( t =0) and refed with serum. Cells were fixed at 30 and 60 min following serum addition, and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1 Bethyl Lab, panels 1) and human Pol-I largest subunit A194 (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panel 4). Scale bar, 5 μM. ( b ) ChIP assays were performed using antibodies specific to human KDM4A (Ab1, Bethyl Lab) and chromatin isolated from starved and starved–refed cells (30 and 60 min after serum addition) and analysed as in to determine KDM4A binding to rDNA promoter (Pr), transcribed region (TrR) or non-transcribed region (nTrR). The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( c ) ChIP assays were performed using antibodies specific to human KDM4A as in b and chromatin immunoprecipitated from starved and starved–refed cells (60 min after serum addition) were analysed by qPCR for the presence either CDC6 or P0 or GAPDH promoters representing a positive and negative controls of the anti-KDM4A ChIP, respectively. The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3.

Techniques: Immunofluorescence, Staining, Isolation, Binding Assay, Negative Control, Immunoprecipitation

( a ) U2OS cells were electroporated with an expression vector encoding either wild-type HA-tagged KDM4A (WT) or catalytically inactive mutant (MUT) or with empty vector (vector) together with a siRNA targeting the 5′UTR of KDM4A, to knockdown endogenous KDM4A while allowing expression of exogenous KDM4A proteins. Cells were grown for 24 h and then starved for another 24 h before serum refeeding in the presence of 5-FUrd. 30 min after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to HA-tag to detect overexpressed proteins, (panels 1), 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panels 4). Bar=5 μM. ( b ) Quantification of 5-FUrd staining shown in a , done as in . Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are significantly different ( P value=4.97 × 10 −9 , Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were electroporated with an expression vector encoding either wild-type HA-tagged KDM4A (WT) or catalytically inactive mutant (MUT) or with empty vector (vector) together with a siRNA targeting the 5′UTR of KDM4A, to knockdown endogenous KDM4A while allowing expression of exogenous KDM4A proteins. Cells were grown for 24 h and then starved for another 24 h before serum refeeding in the presence of 5-FUrd. 30 min after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to HA-tag to detect overexpressed proteins, (panels 1), 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Merged images are shown (panels 4). Bar=5 μM. ( b ) Quantification of 5-FUrd staining shown in a , done as in . Note that the 5-FUrd levels of the cell populations overexpressing the MUT and the WT are significantly different ( P value=4.97 × 10 −9 , Wilcoxon test). The median values are shown as horizontal lines, outliers are shown as open circles; n =3.

Techniques: Expressing, Plasmid Preparation, Mutagenesis, Immunofluorescence, Staining

( a ) Chromatin was isolated from starved and starved–refed cells (30 min after serum addition) and subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution chromatin was subjected to the second IP round using antibody specific to KDM4A and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( b , c ) Chromatin was isolated from starved ( b ) and starved–refed U2OS cells (1 h after serum addition) ( c ) and the same quantities of a chromatin were subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution, the chromatin was subjected for the second IP round using antibodies specific to histone H3, H3K9me1, H3K9me2 and H3K9me3 and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) normalized to the specific H3 signal (representing the difference between the signals from H3 specific antibody and an appropriate IgG, see for raw data) and expressed as % from total chromatin input. Standard deviations from three independent experiments are shown (see for raw data); n =3. ( d , e ). U2OS cells were transfected with KDM4A specific siRNA3. 24 h post-transfection cells were starved for 24 h. Chromatin was isolated from starved ( d ) and starved–refed cells (1 h after serum addition) ( e ). Chromatin was analysed as in b and c (see for raw data); n =3.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) Chromatin was isolated from starved and starved–refed cells (30 min after serum addition) and subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution chromatin was subjected to the second IP round using antibody specific to KDM4A and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) expressed as % from total chromatin input (see for raw data). Standard deviations from three independent experiments are shown; n =3. ( b , c ) Chromatin was isolated from starved ( b ) and starved–refed U2OS cells (1 h after serum addition) ( c ) and the same quantities of a chromatin were subjected for the first round of immunoprecipitation using antibody specific to Pol-I subunit A135. After elution, the chromatin was subjected for the second IP round using antibodies specific to histone H3, H3K9me1, H3K9me2 and H3K9me3 and analysed by qPCR as in . The value of each bar represents the difference between the signals from the specific antibody and from the negative control (an appropriate IgG) normalized to the specific H3 signal (representing the difference between the signals from H3 specific antibody and an appropriate IgG, see for raw data) and expressed as % from total chromatin input. Standard deviations from three independent experiments are shown (see for raw data); n =3. ( d , e ). U2OS cells were transfected with KDM4A specific siRNA3. 24 h post-transfection cells were starved for 24 h. Chromatin was isolated from starved ( d ) and starved–refed cells (1 h after serum addition) ( e ). Chromatin was analysed as in b and c (see for raw data); n =3.

Techniques: Isolation, Immunoprecipitation, Negative Control, Transfection

( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with MAPK inhibitor PD98059 (PD), mTOR inhibitor rapamycin (Rapa), or PI3K inhibitor LY294002 (LY) or a mixture of mTOR and MAPK inhibitors (PD+Rapa) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PI3K inhibitor PI-103, AKT inhibitor AKTVIII (AKT-i), or DMSO (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). ( c ) Quantification of 5-FUrd staining shown in a and b done as in . The effect of inhibitors or combination of inhibitors are all significant ( P value<10 4 , Wilcoxon test), except treatment with PD alone. Note that no incorporation could be measured upon AKT inhibition. The median values are shown as horizontal lines, outliers are shown as open circles; n =3. ( d ) U2OS cells were transfected with an expression vector encoding c-myc-tagged PTEN either wild-type (PTEN-WT) or mutated for its lipid phosphatase activity (PTEN-m). 24 h following transfection, cells were serum starved for 24 h and refed with serum containing 5-FUrd. Cells were fixed and analysed by indirect immunofluorescence using antibodies specific to c-myc Tag to detect overexpressed PTEN (panels 1); KDM4A (panels 2); nuclear DNA was stained by DAPI (panels 3).

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with MAPK inhibitor PD98059 (PD), mTOR inhibitor rapamycin (Rapa), or PI3K inhibitor LY294002 (LY) or a mixture of mTOR and MAPK inhibitors (PD+Rapa) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PI3K inhibitor PI-103, AKT inhibitor AKTVIII (AKT-i), or DMSO (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors. Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (Ab1, panels 1) and 5-FUrd (panels 2); nuclear DNA was stained by DAPI (panels 3). ( c ) Quantification of 5-FUrd staining shown in a and b done as in . The effect of inhibitors or combination of inhibitors are all significant ( P value<10 4 , Wilcoxon test), except treatment with PD alone. Note that no incorporation could be measured upon AKT inhibition. The median values are shown as horizontal lines, outliers are shown as open circles; n =3. ( d ) U2OS cells were transfected with an expression vector encoding c-myc-tagged PTEN either wild-type (PTEN-WT) or mutated for its lipid phosphatase activity (PTEN-m). 24 h following transfection, cells were serum starved for 24 h and refed with serum containing 5-FUrd. Cells were fixed and analysed by indirect immunofluorescence using antibodies specific to c-myc Tag to detect overexpressed PTEN (panels 1); KDM4A (panels 2); nuclear DNA was stained by DAPI (panels 3).

Techniques: Incubation, Immunofluorescence, Staining, Inhibition, Transfection, Expressing, Plasmid Preparation, Activity Assay

( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PDK1 inhibitor GSK2334470 (PDK1i), or PI3K inhibitor LY294002 (LY) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with SGK1 inhibitor GSK650394 (SGK1i), or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and inhibitor (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( c ) U2OS cells were electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against SGK1 (siSGK1-1 and siSGK1-2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd. Thirty minutes after serum addition, cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: ( a ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with PDK1 inhibitor GSK2334470 (PDK1i), or PI3K inhibitor LY294002 (LY) or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and the respective inhibitors (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( b ) U2OS cells were serum starved for 24 h. Starved cells were incubated for 30 min with SGK1 inhibitor GSK650394 (SGK1i), or DMSO as a control (Untr) and refed with serum in the presence of 5-FUrd and inhibitor (+serum) or left starved (no serum). Thirty minutes after serum addition cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM. ( c ) U2OS cells were electroporated with either a non-targeting siRNA (siScr) or two siRNAs directed against SGK1 (siSGK1-1 and siSGK1-2) and kept in serum-free medium for further 24 h before serum refeeding in the presence of 5-FUrd. Thirty minutes after serum addition, cells were fixed and analysed by indirect immunofluorescence using antibodies specific to human KDM4A (panels 1) and Pol-I (panels 2); nuclear DNA was stained by DAPI (panels 3). Scale bar, 5 μM.

Techniques: Incubation, Immunofluorescence, Staining

Effect of the inhibition of various serum-activated kinases on the activation of rDNA transcription and on the nucleolar localization of KDM4A.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: Effect of the inhibition of various serum-activated kinases on the activation of rDNA transcription and on the nucleolar localization of KDM4A.

Techniques: Inhibition, Activation Assay, Over Expression

PI3K–Akt, mTOR and MAPK pathways regulate rRNA gene transcription by targeting RNA Polymerase I machinery (as previously published, see Discussion). PI3K–PDK1–SGK1 signalling cascade regulates activation of rRNA gene transcription by controlling histone modifications at rDNA loci through KDM4A localization. SGK1 may in addition target Pol-I transcription machinery independently of KDM4A.

Journal: Nature Communications

Article Title: The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability

doi: 10.1038/ncomms10174

Figure Lengend Snippet: PI3K–Akt, mTOR and MAPK pathways regulate rRNA gene transcription by targeting RNA Polymerase I machinery (as previously published, see Discussion). PI3K–PDK1–SGK1 signalling cascade regulates activation of rRNA gene transcription by controlling histone modifications at rDNA loci through KDM4A localization. SGK1 may in addition target Pol-I transcription machinery independently of KDM4A.

Techniques: Activation Assay

Journal: Science signaling

Article Title: Epigenetic Activation of AP-1 Promotes Squamous Cell Carcinoma Metastasis

doi: 10.1126/scisignal.2003884

Figure Lengend Snippet: Table 2

Article Snippet: The slides were stained with polyclonal antibodies against KDM4A (1:100; Bethyl), JUN (1:200; Cell Signaling Technology), and FOSL1 (1:100; Santa Cruz Biotechnology) or control IgG (Santa Cruz Biotechnology) at 4°C overnight.

Techniques:

Review

Structured Review

Images

1) Product Images from "Deciphering the epigenetic role of KDM4A in pancreatic β-like cell differentiation from iPSCs"

Similar Products

Image Search Results