Journal: American Journal of Cancer Research

Article Title: Repression of GCN5 expression or activity attenuates c-MYC expression in non-small cell lung cancer

doi:

Figure Lengend Snippet: Quantitative RT-PCR Primers. A list of target genes along with respective forward and reverse primer sequences

Article Snippet: Antibodies used for immunoblot analyses were: anti-ADA2B (Abcam, #57953), anti-β-Actin (Santa Cruz, #sc47778), anti-c-Myc (Cell Signaling Technology, #5605 and #9402), anti-GCN5 (Cell Signaling Technology, #3305), anti-H3 (Abcam, #1791), anti-H3-K9Ac (Abcam, #32129), and anti-PCAF (Cell Signaling Technology, #3378).

Techniques: Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: Identification of KAT2A as a host factor promoting cccDNA transcription. (A) The reported succinyltransferases and desuccinylases were summarised in the table. (B) After 4 days of siRNA treatment, total RNA was extracted by TRNzol Universal reagent. The silencing efficiency of related genes in Huh-7 cells was analysed by real-time PCR with specific primers. β-actin was used as the internal control. (C) The indicated siRNA (50 pmol) and monomeric linearised HBV DNA (1 μg) were co-transfected into huh-7 cells by using Lipofectamine™ 3000 Transfection Reagent. The supernatant was collected and HBeAg and HBsAg levels were detected by ELISA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A knockdown suppresses cccDNA transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post shRNA transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) Northern blotting and real-time PCR were used to analyse the effect of KAT2A knockdown on HBV RNAs. (C) HBV cccDNA was extracted by a modified Hirt DNA extraction protocol and then Southern blotting and Taq-man probe qPCR were used to detect the HBV cccDNA level. The mitochondrial gene Cox1 was hybridised as the loading control for HBV cccDNA. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBV core protein (HBc) was produced by HBV infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Western blot was used to detect the level of HBsAg in cells (left). Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA (right). Cox1, cyclooxygenase 1; rRNA, ribosomal RNA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Infection, shRNA, Transduction, Expressing, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Modification, DNA Extraction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A overexpression promotes HBV transcription in the HBV infection cell model. HepG2-NTCP cells were infected HBV particles at 1-day post lentivirus expressing KAT2A plasmids transduction. At 4 days after infection. (A) The expression of KAT2A was detected by western blot. (B) The effect of KAT2A overexpression on HBV RNAs was analysed by Northern blotting and real-time PCR. (C) Taq-man probe qPCR was used to detect the HBV cccDNA level. (D) The level of HBV 3.5-kb RNA, total HBV RNAs, and cccDNA were used to calculate the ratio of HBV 3.5-kb RNA to cccDNA and total HBV RNAs to cccDNA. (E) HBV core DNA was quantified by Southern blotting and absolute quantification PCR. The level of β-actin was used as a loading control for HBV core DNA. (F) HBc was produced by HBV-infected HepG2-NTCP cells. Intracellular HBc was visualised by immunofluorescence staining. The scale bar is 100 μm. (G) Cell culture supernatant was collected for HBsAg and HBeAg analysis via ELISA. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Over Expression, Infection, Expressing, Transduction, Western Blot, Northern Blot, Real-time Polymerase Chain Reaction, Southern Blot, Produced, Immunofluorescence, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A bound to cccDNA and regulated H3K79 succinylation on the cccDNA minichromosome. (A) HepG2-NTCP cells were infected with HBV particles for 4 days, HBV core protein and endogenous KAT2A were observed by immunofluorescence assay using the specific antibodies. The scale bar is 10 μm. (B) Cross-linked chromatin from the HBV-infected and non-infected HepG2-NTCP nucleus was immunoprecipitated with a specific antibody or the control lgG. Taq-man probe qPCR was used to detect the HBV cccDNA level. ChIP results are expressed as% of input. (C–E) HepG2-NTCP cells were infected with HBV particles at 1-day post shKAT2A transduction. On 4 days post-infection. (C) The levels of H3ac, H3K9ac, and H3K14ac associated with cccDNA, GAPDH, or MYH6 promoter were analysed by ChIP assay with anti-H3ac, anti-H3K9ac, anti-H3K14ac, and the corresponding IgG, respectively. (D) ChIP assay was performed with anti-Pan-succ antibody. (E) The levels of H3K79succ and H3K122succ associated with cccDNA, GAPDH, or MYH6 promoter were analysed by ChIP assay with anti-H3K79succ, anti-H3K122succ, and the corresponding lgG, respectively. (F) ChIP-Seq analysis of the H3K79succ modification on the cccDNA minichromosome. The HBV-specific reads were quantified and normalised by the reads mapped to the human genome. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Infection, Immunofluorescence, Immunoprecipitation, Transduction, ChIP-sequencing, Modification

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: KAT2A binds to the cccDNA minichromosome through interaction with HBc. (A) HepG2-NTCP cells were co-transfected with plasmids encoding KAT2A and Flag-HBc for 3 days, the cells were subjected to Co-IP assay with the indicated antibody. The expression of the indicated proteins was analysed by western blot. (B,C) HepG2-NTCP cells were transduced with lentivirus expressing KAT2A or shKAT2A for 24 h, then infected with HBV wild type virus (HBV WT virus) and HBc-deficient virus (HBV-ΔHBc virus) for 4 days. Total HBV RNAs and cccDNA were extracted and quantified by real-time PCR and Taq-man probe qPCR for calculating the ratio of total HBV RNAs to cccDNA. The HBc proteins were detected by immunoblotting analysis. (D) HepG2-NTCP cells in 100 mm dishes were infected by HBV WT virus and HBV-ΔHBc virus. The cells were used for ChIP assays with the anti-KAT2A antibody. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Transfection, Co-Immunoprecipitation Assay, Expressing, Western Blot, Transduction, Infection, Real-time Polymerase Chain Reaction

Journal: Frontiers in Microbiology

Article Title: KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome

doi: 10.3389/fmicb.2021.795388

Figure Lengend Snippet: Antiviral activity of KAT2A knockdown in vivo . (A) Schematic depiction of experiments in C57BL/6 mice. (B) Serum HBV DNA was detected by absolute quantification PCR. (C) Serum HBsAg was measured via ELISA. (D) HBV 3.5-kb RNA and total HBV RNAs in liver tissue were measured by real-time PCR. (E) HBV cccDNA in liver tissue was detected by Taq-man probe qPCR. (F) The level of HBV DNA in liver tissue was detected by absolute quantification PCR. (G) HBc in liver tissues was analysed by IHC. The scale bar is 50 μm. (H,I) The levels of H3K9ac, H3K14ac associated with cccDNA were analysed by ChIP assay. (J) The level of H3K79succ associated with cccDNA was analysed by ChIP assay. * P < 0.05.

Article Snippet: pCMV6-XL5-KAT2A was purchased from OriGene (SC125929).

Techniques: Activity Assay, In Vivo, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Acetylation of C/EBPα inhibits its granulopoietic function

doi: 10.1038/ncomms10968

Figure Lengend Snippet: ( a ) Western blot analysis with anti-pan-acetyl-lysine antibody following immunoprecipitation (IP) of C/EBPα with rabbit anti-C/EBPα in HL-60 and Molm-14 human myeloid cell lysates. Rabbit anti-GFP antibody was used as IP control. ( b , c ) GCN5 decreases the ability of C/EBPα to transactivate a minimal p(CEBP) 4 TK promoter in a dose-dependent manner and is dependent on the GCN5 histone acetyltransferase (HAT) domain. 293T cells were transiently transfected with p(CEBP) 4 TK, pRL-null, and pcDNA6 expression plasmids for C/EBPα and with GCN5 or GCN5 (-HAT), respectively. Protein expression of corresponding constructs were shown in . Luciferase activity was measured in duplicate for each experiment and data are shown as mean±s.d. ( N =3). ( d ) Knockdown of endogenous GCN5 results in an increase in C/EBPα transactivation potential. GCN5 knockdown in 293T cells enhanced C/EBPα transactivation capacity on a minimal p(CEBP) 4 TK promoter with pRL-null and pcDNA6 C/EBPα plasmids. Firefly luciferase readings were normalized against internal control renilla luciferase. Luciferase activity was measured in duplicate for each experiment and data are shown as mean±s.d. ( N =3). ( e ) Western blot demonstrating knockdown efficiencies of endogenous GCN5 by 3 different shRNAs in 293T cells used in d . ( f ) GCN5 acetylates C/EBPα at K298, K302 and K326. C/EBPα peptides were incubated with the GST-HAT domain of GCN5 in the presence of 3 H-labeled acetyl-CoA. Tritium incorporation by C/EBPα peptides was measured by scintillation counting. Error bars represent mean±s.e.m. Experiments were performed twice in duplicate. ( g ) Structure of the C/EBPα basic region-leucine zipper domain bound to DNA. Arrows indicate the location of the acetylated lysine residues. ( h ) Endogenous C/EBPα is acetylated in the basic leucine zipper region in HL-60 and Molm-14 cells. C/EBPα protein was immunoprecipitated using a rabbit antibody recognizing total C/EBPα protein, followed by immunoblotting against acetylated C/EBPα antibodies (K298, K302, and K326). Rabbit anti-GFP antibody was used as a control for IP. ** P <0.01 and *** P <0.001; Student's unpaired t -test ( b , d and f ).

Article Snippet: GST-tagged recombinant GCN5 (Catalogue #K311-381G) was also purchased from SignalChem.

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Construct, Luciferase, Activity Assay, Incubation, Labeling