Journal: PLOS ONE

Article Title: Characterization of a KDM5 small molecule inhibitor with antiviral activity against hepatitis B virus

doi: 10.1371/journal.pone.0271145

Figure Lengend Snippet: Biochemical potency and selectivity of GS-080 against KDM enzymes.

Article Snippet: KDM primer/probe sets used for qRT-PCR were from Thermo Fisher (4392420): Hs00231908_m1, Hs00981910_m1, Hs01011846_m1, Hs00190491_m1, Hs00206360_m1, Hs00392119_m1, Hs00323906_m1, Hs00250616_s1, Hs00988859_s1, and Hs01096550_m1.

Techniques: Concentration Assay

Journal: PLOS ONE

Article Title: Characterization of a KDM5 small molecule inhibitor with antiviral activity against hepatitis B virus

doi: 10.1371/journal.pone.0271145

Figure Lengend Snippet: KDM4 and KDM5 transcripts were depleted by siRNA either individually ( KDM4A , B , C , D , or E ; KDM5A , B , C , or D ) or simultaneously ( KDM4A – E pool; KDM5A – D pool) in HBV-infected PHH. DDB1 , the well-characterized HBV host restriction factor, was also depleted by siRNA for comparison. Fourteen days after initiation of siRNA transfection, levels of (A) intracellular HBV RNA, (B) secreted HBeAg, and (C) secreted HBsAg were measured by qRT-PCR or immunoassay, respectively, for each siRNA condition. (D) The kinetics of HBeAg inhibition is shown for KDM4A – E pool, KDM5A – D pool, and DDB1 siRNA conditions. Data shown are the average of two biological replicate experiments and error bars represent the standard deviation.

Article Snippet: KDM primer/probe sets used for qRT-PCR were from Thermo Fisher (4392420): Hs00231908_m1, Hs00981910_m1, Hs01011846_m1, Hs00190491_m1, Hs00206360_m1, Hs00392119_m1, Hs00323906_m1, Hs00250616_s1, Hs00988859_s1, and Hs01096550_m1.

Techniques: Infection, Comparison, Transfection, Quantitative RT-PCR, Inhibition, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Kdm4b Histone Demethylase Is a DNA Damage Response Protein and Confers a Survival Advantage following γ-Irradiation *

doi: 10.1074/jbc.M113.491514

Figure Lengend Snippet: Kdm4b- and KDM4D-EGFP recruit to DNA damage. U2OS osteosarcoma cells were transfected with Kdm4b-EGFP. 24 h after transfection 2-photon laser micro-irradiation was used to measure the recruitment of EGFP-tagged Kdm4 demethylases to DNA damage. A, both Kdm4b-EGFP and KDM4D-EGFP recruited readily to the DNA damage tracks. Graphs represent the mean values for 18, 30, 25, and 16 nuclei ± S.E. (Kdm4a–c, respectively). B, Kdm4a-EGFP also recruited to the DNA damage tracks but at later time points. C, representative images show the accumulation of Kdm4b-EGFP (C, top) and KDM4D-EGFP (C, bottom) on the DNA damage tracks.

Article Snippet: Kdm4a, Kdm4c, and KDM4D Gateway donor vectors were constructed using clones from OriGene (Rockville, MD) as template cDNA.

Techniques: Transfection, Irradiation

Journal: The Journal of Biological Chemistry

Article Title: Kdm4b Histone Demethylase Is a DNA Damage Response Protein and Confers a Survival Advantage following γ-Irradiation *

doi: 10.1074/jbc.M113.491514

Figure Lengend Snippet: Kdm4-EGFP does not accumulate at IRIF. U2OS cells were plated on glass coverslips and transfected with EGFP-tagged Kdm4a (A), Kdm4b (B), Kdm4c (C), or KDM4D (D). 24 h after transfection, cells were treated with 2 Gy of γ-irradiation and fixed at 60 min post-irradiation. Cells were stained for γH2AX, and the nuclei were counterstained with DAPI. Line scans show the fluorescence intensity for the EGFP-tagged Kdm4 protein (gray line, curve area filled) and the Cy3-stained γH2AX (black line). The line scans shown were performed on the lines indicated in the Kdm4-EGFP panels (delineated by arrows).

Article Snippet: Kdm4a, Kdm4c, and KDM4D Gateway donor vectors were constructed using clones from OriGene (Rockville, MD) as template cDNA.

Techniques: Transfection, Irradiation, Staining, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: HPV11 targeting KDM4A regulates the polarization of macrophage M 1 and promotes the development of nasal inverted papilloma

doi: 10.1186/s12964-024-01971-6

Figure Lengend Snippet: The primers for qRT-PCR

Article Snippet: Primary antibodies used were: anti- KDM4A (1:5000, Proteintech, Wuhan, China), anti-HPV11E6 (1:1000, MyBioSource, San Diego, USA), anti-HPV11E7 (1:1000, Abcam, Shanghai, China), anti-CD206 (1:1000, Proteintech, Wuhan, China), anti-CD86 (1:2000, Proteintech, Wuhan, China), anti-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-IκB-α (1:10000, Proteintech, Wuhan, China) and anti-GAPDH (1:20000, Proteintech, Wuhan, China).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: HPV11 targeting KDM4A regulates the polarization of macrophage M 1 and promotes the development of nasal inverted papilloma

doi: 10.1186/s12964-024-01971-6

Figure Lengend Snippet: A Pupil map of integration sites of HPV ( +) viral genes in the human chromosome genome in NIP: each short band marked on the human chromosome in the figure indicates an integration event, and the high-frequency integration site KDM4A for HPV infection was identified. B-D The mRNA expression of HPV11E6, HPV11E7, and KDM4A in the cells of each group was verified by qRT-PCR. E , F The protein expression of HPV11E6, HPV11E7, and KDM4A in cells of each group was verified by Western blot. G - I Image J was used to analyze the gray values of each strip. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.002, * P < 0.033, ns means no statistical significance ( P > 0.12)

Article Snippet: Primary antibodies used were: anti- KDM4A (1:5000, Proteintech, Wuhan, China), anti-HPV11E6 (1:1000, MyBioSource, San Diego, USA), anti-HPV11E7 (1:1000, Abcam, Shanghai, China), anti-CD206 (1:1000, Proteintech, Wuhan, China), anti-CD86 (1:2000, Proteintech, Wuhan, China), anti-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-IκB-α (1:10000, Proteintech, Wuhan, China) and anti-GAPDH (1:20000, Proteintech, Wuhan, China).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Stripping Membranes

Journal: Cell Communication and Signaling : CCS

Article Title: HPV11 targeting KDM4A regulates the polarization of macrophage M 1 and promotes the development of nasal inverted papilloma

doi: 10.1186/s12964-024-01971-6

Figure Lengend Snippet: Overexpression of HPV11E6/E7 can promote cell proliferation and migration in vitro, and KDM4A gene knockout can inhibit cell proliferation and migration caused by HPV11E6/E7 overexpression. A Cell proliferation was measured by the CCK-8 method. B , C Colony formation assay was used to detect cell proliferation. D , E EdU fluorescent labeling was used to detect cell proliferation. F , G Transwell assay was used to detect cell migration. H , I Wound healing test to detect cell migration. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.002, * P < 0.033

Article Snippet: Primary antibodies used were: anti- KDM4A (1:5000, Proteintech, Wuhan, China), anti-HPV11E6 (1:1000, MyBioSource, San Diego, USA), anti-HPV11E7 (1:1000, Abcam, Shanghai, China), anti-CD206 (1:1000, Proteintech, Wuhan, China), anti-CD86 (1:2000, Proteintech, Wuhan, China), anti-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-IκB-α (1:10000, Proteintech, Wuhan, China) and anti-GAPDH (1:20000, Proteintech, Wuhan, China).

Techniques: Over Expression, Migration, In Vitro, Gene Knockout, CCK-8 Assay, Colony Assay, Labeling, Transwell Assay

Journal: Cell Communication and Signaling : CCS

Article Title: HPV11 targeting KDM4A regulates the polarization of macrophage M 1 and promotes the development of nasal inverted papilloma

doi: 10.1186/s12964-024-01971-6

Figure Lengend Snippet: Overexpression of HPV11E6/E7 can promote M 1 polarization of macrophages under co-culture conditions, and knockout of KDM4A can reduce the degree of M 1 polarization of macrophages. A - C Typical markers of M 1 macrophages (CD206, IL-6, IL-1β) were detected by qRT-PCR. D - F Typical markers of M 2 macrophages (CD206, IL-10, Arg-1) were detected by qRT-PCR. G - K Western blot was used to detect the expression levels of CD86 and CD206 proteins and the expressions of NF-κB P65, Phospho-NF-κB P65, and Phospho-IκB proteins in the M 1 macrophage polarization pathway NF-kB. The internal parameter of CD86, CD206 and Phospho-IκB is GAPDH, and the internal parameter of Phospho-NF-κB P65 is NF-κB P65. *** P < 0.001, ** P < 0.002, * P < 0.033, ns means no statistical significance( P > 0.12)

Article Snippet: Primary antibodies used were: anti- KDM4A (1:5000, Proteintech, Wuhan, China), anti-HPV11E6 (1:1000, MyBioSource, San Diego, USA), anti-HPV11E7 (1:1000, Abcam, Shanghai, China), anti-CD206 (1:1000, Proteintech, Wuhan, China), anti-CD86 (1:2000, Proteintech, Wuhan, China), anti-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-IκB-α (1:10000, Proteintech, Wuhan, China) and anti-GAPDH (1:20000, Proteintech, Wuhan, China).

Techniques: Over Expression, Co-Culture Assay, Knock-Out, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: HPV11 targeting KDM4A regulates the polarization of macrophage M 1 and promotes the development of nasal inverted papilloma

doi: 10.1186/s12964-024-01971-6

Figure Lengend Snippet: In vivo experiments have shown that overexpression of HPV11E6/E7 can promote subcutaneous tumor formation in nude mice, and KDM4A knockout can inhibit the proliferation of subcutaneous tumor tissue. Overexpression of HPV11E6/E7 can promote local recruitment of more macrophages and induce M 1 -type polarization of macrophages in subcutaneous tumors of nude mice. Knockout of KDM4A gene can reduce the number of macrophages recruited and the degree of M 1 -type polarization of macrophages. A , B The volume of subcutaneous mass tissue in each group in the nude mouse model. C - E The expression level of HPV11E6, HPV11E7, and KDM4A in subcutaneous mass tissues of mice in each group was verified by qRT-PCR. F H&E staining of subcutaneous tumor tissue. G Tissue immunofluorescence F4/80 + CD86 double staining. H Tissue immunofluorescence F4/80 + CD206 double staining. I-K Semi-quantitative analysis of the average fluorescence intensity of F4/80, CD86, and CD206 in each group. Data are presented as mean ± SD; *** P < 0.001, ** P < 0.002, ns means no statistical significance( P > 0.12)

Article Snippet: Primary antibodies used were: anti- KDM4A (1:5000, Proteintech, Wuhan, China), anti-HPV11E6 (1:1000, MyBioSource, San Diego, USA), anti-HPV11E7 (1:1000, Abcam, Shanghai, China), anti-CD206 (1:1000, Proteintech, Wuhan, China), anti-CD86 (1:2000, Proteintech, Wuhan, China), anti-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-NF-κB P65 (1:5000, Proteintech, Wuhan, China), anti-Phospho-IκB-α (1:10000, Proteintech, Wuhan, China) and anti-GAPDH (1:20000, Proteintech, Wuhan, China).

Techniques: In Vivo, Over Expression, Knock-Out, Expressing, Quantitative RT-PCR, Staining, Immunofluorescence, Double Staining, Fluorescence

Journal: Nucleic Acids Research

Article Title: Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops

doi: 10.1093/nar/gku823

Figure Lengend Snippet: LSD1 and JMJD2A upon RA exposure are recruited to the CASP9 promoter and RARE sites and are essential for CASP9 induction by RA and methylation–demethylation cycle. ( a, b ) Recruitment of LSD1 and JMJD2A to the promoter and RARE of CASP9 gene. MCF7 cells were serum starved and exposed to 300-nM RA at the indicated times (15, 30, 60 and 240 min). qChIP was carried out using specific antibodies recognizing LSD1 and JMJD2A. The panels show the time course of the recruitment of LSD1 (a) and JMJD2A (b) to the RARE and promoter sequences of CASP9 analyzed by qPCR. The black, horizontal line in each plot indicates the percent of input from a control ChIP (Ab: non-immune serum). ( c, d ) LSD1 and JMJD2A depletion inhibits RA-induced transcription of CASP9 . MCF7 were transiently transfected with LSD1 siRNA or JMJD2A siRNA. After 48 h, total RNA was prepared from control cells (starved) or RA-induced cells (300-nM RA for 45 min) and analyzed by qPCR with specific primers to CASP9 mRNA. The statistical analysis derives from at least three experiments in triplicate ( n ≥ 9; mean ± SD); * P < 0.01 (matched pairs t- test) compared to RA-unstimulated sample; ** P < 0.01 (student t- test): comparison between SCR and specific siRNAs; °° P < 0.01 (student t- test): comparison between promoter and RARE regions. ( e ) Depletion of LSD1 or JMJ2DA inhibits the methylation changes induced by RA. qChIP was performed on cells transfected with LSD1 siRNA or JMJD2A siRNA and induced with 300-nM RA for 15 min. qChIP was carried out using specific antibodies recognizing H3K4me3, H3K4me2, H3K9me3 and H3K9me2. The statistical analysis derives from at least three experiments in triplicate ( n ≥ 9; mean ± SD); * P < 0.01 (matched pairs t- test) compared to RA-unstimulated sample; ** P < 0.01 (student t- test): comparison between SCR and specific siRNAs. Transfection efficiency was monitored by FACS (Alexa Fluor or co-transfected pEGFP Vector).

Article Snippet: To rescue JMJD2A activity, cells were transfected with pCMV6-AC-GFP plasmid containing JMJD2A full-length (RG200574, OriGene Technologies, Inc.).

Techniques: Methylation, Transfection, Plasmid Preparation

Journal: Clinical and Molecular Hepatology

Article Title: Glutamate dehydrogenase 1-dependent α-ketoglutarate promotes hepatitis B virus transcription by modulating histone methylations on the covalently closed circular DNA minichromosome

doi: 10.3350/cmh.2024.0694

Figure Lengend Snippet: GDH1-derived αKG interacts with KDM4A and downregulates histone methylation of H3K4, H3K9 and H4K20 on cccDNA minichromosome. (A) HBV-infected HepG2-NTCP cells were transduced with lentivirus-expressed shGDH1 for 5 days to measure KDMs demethylase activity. (B) HBV-infected cells transduced with GDH1-expressing lentivirus subjected to detect KDM4A demethylase activity. (C, D) The interaction between KDM4A and GDH1 was detected by immunoprecipitation and PLA in HepG2-NTCP cells. (E, F) MST analysis was performed by using Monolith NT.115 (NanoTemper Technologies). MST traces for single dose of αKG (right panel) and dose response curve (left panel) for KDM4A WT (E) and KDM4A mut (F) were provided. (G) ChIP assay revealed the enrichment of KDM4A, KDM1A, KDM1B on cccDNA, GAPDH , MYH6 promoter. (H) HBV-infected cells transduced with GDH1 or treated with DM-αKG, ChIP assay showed the enrichment of H3K4me3, H3K9me3, H4K20me3 on cccDNA, GAPDH , MYH6 promoters. (I) Localization of H3K4me3, H3K9me3, H4K20me3 along the HBV genome was exhibited by using ChIP-Seq. ** P <0.01; *** P <0.001. GDH1, glutamate dehydrogenase 1; αKG, α-ketoglutarate; cccDNA, covalently closed circular DNA; HBV, hepatitis B virus; KDMs, histone lysine demethylases; PLA, proximity ligation assay; MST, micro scale thermophoresis; WT, wild-type; ChIP, chromatin immunoprecipitation; DM-αKG, dimethyl-αKG; LSD, lysine-specific histone demethylase.

Article Snippet: Assay was performed by using JMJD2A (KDM4A) Homogenous Assay Kit (#50413; BPS Bioscience, Milan, Italy) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Methylation, Infection, Transduction, Activity Assay, Expressing, Immunoprecipitation, ChIP-sequencing, Virus, Proximity Ligation Assay, Chromatin Immunoprecipitation