Journal: Journal of Biological Chemistry

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes

doi: 10.1074/jbc.m113.538777

Figure Lengend Snippet: FIGURE 8. PLZF-RAR represses transcription of CDKN1A epigenetically by histone deacetylation and DNA methylation. A, structure of the human CDKN1A gene promoter. The arrows indicate the locations of the qChIP-PCR primer binding sites. B, qChIP assays showing PLZF-RAR binding at the endog- enous CDKN1A proximal promoter using an antibody against RAR. Cells were transfected with the PLZF-RAR expression vector and immunoprecipitated (IP) with an anti-RAR antibody. IgG, control ChIP antibody. C and D, qChIP-PCR assays showing histone modifications at the endogenous CDKN1A proximal promoter. Cells were transfected with the PLZF-RAR expression vector and lysates were immunoprecipitated with the indicated antibodies (IgG, Ac-H3, Ac-H4,H3K4-Me3,orH3K9-Me3).E,Me-DIP(methylatedDNAChIP)assaysshowingincreasedDNAmethylationattheendogenousCDKN1Aproximalpromoter following ectopic PLZF-RAR expression. HEK293 cells were transfected with a PLZF-RAR expression vector, lysates were immunoprecipitated (IP) with the antibody recognizing methylated DNA, and precipitated DNA was amplified using the primers indicated in A. F, bisulfite sequencing analysis of the methylated CDKN1Apromoter.TheproximalpromotersequencesofCDKN1A,withpotentiallymethylatedCpGsitesareshowningrayovals,andtheSp1bindingGC-boxes shown above. Open ovals, unmethylated CpG; filled ovals, methylated CpG. G, co-immunoprecipitation of PLZF-RAR, MBD3, NuRD (MTA2), DNMT1, DNMT3b, and HP1. Cell lysates from either HEK293 cells transfected with a pcDNA3 vector or a PLZF-RAR expression vector were immunoprecipitated (IP) using anti-PLZF, anti-RAR or anti-IgG antibodies and analyzed by Western blot (WB) with the indicated antibodies. H, qChIP assays showing the proximal promoter binding of PLZF-RAR, MBD3, the NuRD-HDAC3 complex, DNMT1/3b and HP1 proteins in HEK293 cells transfected with the PLZF-RAR expression vector. *, p 0.05; N.S., not significant; t test.

Article Snippet: Antibodies against p21, p53, HDAC1, HDAC3, MDM2, PLZF, RAR , Sp1, GAPDH, Myc tag, Ac-H3, Ac-H4, H3K4-Me3, H3K9Me3, MBD3, HP1, MTA2, DNMT1, DNMT3b, mSin3A, NCoR, and SMRT were purchased from Upstate, Chemicon, Cell Signaling Technology, Abcam, Calbiochem, and Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, Binding Assay, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Control, Methylation, Amplification, Methylation Sequencing, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Promyelocytic Leukemia Zinc Finger-Retinoic Acid Receptor α (PLZF-RARα), an Oncogenic Transcriptional Repressor of Cyclin-dependent Kinase Inhibitor 1A (p21WAF/CDKN1A) and Tumor Protein p53 (TP53) Genes

doi: 10.1074/jbc.m113.538777

Figure Lengend Snippet: FIGURE 9. PLZF-RAR stimulates cell proliferation and represses CDKN1A transcription in HL-60 cells through inhibitory histone modifications and DNA methylation. A, MTT assay of cell proliferation. HL-60 cells transfected with either pcDNA3 or pSG5-PLZF-RAR plasmid were grown for 1–4 days and analyzed for MTT to formazan conversion using colorimetry at 540–600 nm. B and C, Western blot (WB) and RT-qPCR analysis of protein and mRNA levels of the HL-60cellstransfectedwiththePLZF-RARexpressionvectororacontrolplasmid.D–F,qChIPassaysofmodifiedhistonesAc-H3,Ac-H4,H3K4-Me3,H3K9-Me3, and PLZF-RAR binding at the proximal CDKN1A promoter. HL-60 cells were transfected with the PLZF-RAR expression vector and analyzed for changes in Ac-H3, Ac-H4, H3K4-Me3, and H3K9-Me3 levels at the indicated regions (Fig. 8A) using the indicated antibodies. G, Me-DIP assays to assess DNA methylation of the endogenous CDKN1A proximal promoter in HL-60 cells transfected with PLZF-RAR expression vector. Alpha X1, positive control. H–K, qChIP assays of MBD3, NuRD (MTA2), DNMT1 and -3b, and HP1 binding at the proximal CDKN1A promoter in HL-60 cells transfected with a PLZF-RAR expression vector. *, p 0.05; N.C., negative control; P.C., positive control; N.S., not significant; t test.

Article Snippet: Antibodies against p21, p53, HDAC1, HDAC3, MDM2, PLZF, RAR , Sp1, GAPDH, Myc tag, Ac-H3, Ac-H4, H3K4-Me3, H3K9Me3, MBD3, HP1, MTA2, DNMT1, DNMT3b, mSin3A, NCoR, and SMRT were purchased from Upstate, Chemicon, Cell Signaling Technology, Abcam, Calbiochem, and Santa Cruz Biotechnology.

Techniques: DNA Methylation Assay, MTT Assay, Transfection, Plasmid Preparation, Colorimetric Assay, Western Blot, Quantitative RT-PCR, Binding Assay, Expressing, Positive Control, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Transcription suppression is mediated by the HDAC1–Sin3 complex in Xenopus nucleoplasmic extract

doi: 10.1016/j.jbc.2022.102578

Figure Lengend Snippet: The Sin3 deacetylase complex promotes transcription suppression. A , sequential IP schematic. B , mock, HDAC1, or HDAC2 IPs were performed in NPE. The supernatants from HDAC1 or HDAC2 IPs were then used for a second round of IPs using the converse antibody. Isolated proteins were then analyzed by Western blot with the indicated antibodies (n = 3). 10% of total reaction sample (IN), supernatant (S), pellet (P). C , NPE was immunodepleted using preimmune (ΔMock) or MTA2 (ΔMTA2) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. D , pActin was incubated in mock- or MTA2-depleted extract. RNA was isolated and quantified after 120 min (n = 2). E , NPE was immunodepleted using preimmune (ΔMock) or Sin3a (ΔSin3a) antibodies. Depleted extracts were analyzed by Western blot using the indicated antibodies. F , pActin was incubated in mock- or Sin3a-depleted extract. RNA was isolated and quantified after 120 min (n = 2). G , NPE was immunodepleted using preimmune (ΔMock) or HDAC1 (ΔHDAC1) antibodies. HDAC1-depleted extract was then supplemented with immunoprecipitated proteins recovered by preimmune (+Mock IP) or Sin3a (+Sin3a IP) IP. pActin was incubated in each extract and RNA was isolated and quantified after 120 min (n = 2). Student t test: p -value < 0.05 (∗), p -value < 0.01 (∗∗), p -value < 0.001 (∗∗∗), not significant (n.s.). Error bars represent ±1 SD. IP, immunoprecipitation; NPE, nucleoplasmic extract.

Article Snippet: Commercial antibodies were used to detect Histone H3 (ThermoFisher PA5-16183), H4K5ac (Abclonal A15233), H4K8ac (Abclonal A7258), H4K16ac (Abclonal A5280), H3K27ac (Abclonal A7253), MTA2 (Novus Biologicals NB100-56483SS), CoREST (Millipore 07-455), H2AK5ac (Thermofisher 720070), H2BK20ac (Millipore Sigma 07-347), H3K9/14ac (ThermoFisher 49-1010), H3K23ac (Cell Signaling 8848), and RNAPII (Bethyl Laboratories A300-653A).

Techniques: Histone Deacetylase Assay, Isolation, Western Blot, Incubation, Immunoprecipitation

Journal: Cell Reports

Article Title: SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability

doi: 10.1016/j.celrep.2024.113778

Figure Lengend Snippet: Sin3A prevents fork breakage in stressed conditions (A) Images of cells immunostained for chr-bound RAD51 (green) and FANCD2 (red) proteins. DNA stained with DAPI (blue). Scale bar, 10 μm. siRNAs and HU as indicated. Plots show number of FANCD2 (left) or RAD51 (right) foci per cell. Mean in black. Data are pooled from 3 different assays. >1,500 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. Immunoblot detection of Sin3A. H3, loading control. (B) Images of cells immunostained for γH2AX (red) and chr-bound RPA (green) proteins. DNA stained with DAPI (blue). Scale bar, 25 μm. Treatment as in (A). Histograms show the percentage (mean + SD) of γH2AX (top), chr-bound RPA-positive cells (left), and double-positive cells. n = 3. >400 cells scored per condition and assay. Negative staining determined in untreated control cells. ∗ p = 0.0346 (top) and p = 0.0231 (bottom left); ∗∗ p = 0.0037; unpaired two-tailed Student’s t test. (C) Images of U2OS SEC-C (cells stably expressing Cas9) cells immunostained for γH2AX (red) protein. DNA stained with DAPI (blue). Scale bar, 10 μm. RNA guides (72 h) and HU (3 mM, 4 h) as indicated. Immunoblot detection of Sin3A in indicated samples. GAPDH, loading control. Plot shows distribution of γH2AX intensity values. Median in black. Data are pooled from 2 different assays. >140 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (D) Plot shows distribution of γH2AX intensity values in cells treated (4 h) with HU (3 mM) combined with sodium butyrate (NaB, 5 mM), trichostatin A (TSA, 250 nM), and romidepsin (50 nM). Median in black. Data are pooled from 4, 2, and 3 different assays, respectively. >1,100 cells scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (E) Images of cells immunostained for chr-bound 53BP1 (green) protein. DNA was stained with DAPI (blue). Scale bar, 10 μm. siRNAs and HU as indicated. Plot shows number of 53BP1 foci per cell. Data are pooled from 3 different assays. >1,400 cells were scored per condition. ∗∗∗ p < 0.0001; two-tailed Mann-Whitney test. (F) Representative images of comet assay. Scale bar, 100 μm. siRNAs and HU as indicated. Histogram shows tail moment (mean + SD). n = 3. ∗∗ p = 0.0067; two-tailed unpaired Student’s t test. (G) Same as in (F) in indicated samples. Scale bar, 100 μm. Histogram shows tail moment (mean + SD). n = 4. n.s. p = 0.1087; two-tailed unpaired Student’s t test. Immunoblot detection of MTA2. GAPDH, loading control. siMTA2, MTA2 siRNA-transfected cells. HU (3 mM, 24 h) except for (C) and (D). siRNA transfection (72 h). All replicates are biological replicates. See also Figure S2 .

Article Snippet: Rabbit anti MTA2 , Atlas Antibodies , HPA006214; RRID: AB_1079421.

Techniques: Staining, Two Tailed Test, MANN-WHITNEY, Western Blot, Control, Negative Staining, Stable Transfection, Expressing, Single Cell Gel Electrophoresis, Transfection

Journal: Cell Reports

Article Title: SIN3A histone deacetylase action counteracts MUS81 to promote stalled fork stability

doi: 10.1016/j.celrep.2024.113778

Figure Lengend Snippet:

Article Snippet: Rabbit anti MTA2 , Atlas Antibodies , HPA006214; RRID: AB_1079421.

Techniques: Recombinant, Control, Protease Inhibitor, Imaging, Reverse Transcription, Plasmid Preparation, Software, Magnetic Beads, Blocking Assay, In Situ, Western Blot, Membrane

Journal: Heliyon

Article Title: Mechanism of HIF-1α promoting proliferation, invasion and metastasis of nasopharyngeal carcinoma by regulating MMP-2 in hypoxic microenvironment

doi: 10.1016/j.heliyon.2024.e40760

Figure Lengend Snippet: Primer sequences used in this study.

Article Snippet: 10 % skimmed milk powder (solarbio, China) was sealed at room temperature for 2 h. The primary antibodies HIF-1α (1:1000, Abcam, USA), MMP-2 (1:1000, Cohesion Biosciences, UK), Fas (1:1000, Abcam, USA), BRCA1 (1:1000, Cohesion Biosciences, UK), TIMP-1 (1:1000, Abcam, USA), AKT1 (1:1000, Cohesion Biosciences, UK), VEGFA (1:1000, Abcam, USA), MET (1:1000, Cohesion Biosciences, UK), MMP-9 (1: 1000, Cohesion Biosciences, UK), MTA2 (1:1000, Cohesion Biosciences, UK), and β-actin (1:5000, Cohesion Biosciences, UK) were incubated overnight at 4 °C.

Techniques: Sequencing

Journal: Heliyon

Article Title: Mechanism of HIF-1α promoting proliferation, invasion and metastasis of nasopharyngeal carcinoma by regulating MMP-2 in hypoxic microenvironment

doi: 10.1016/j.heliyon.2024.e40760

Figure Lengend Snippet: Genes up-regulated or down-regulated at least 2.0-fold by silencing HIF-1α in HONE1 cells.

Article Snippet: 10 % skimmed milk powder (solarbio, China) was sealed at room temperature for 2 h. The primary antibodies HIF-1α (1:1000, Abcam, USA), MMP-2 (1:1000, Cohesion Biosciences, UK), Fas (1:1000, Abcam, USA), BRCA1 (1:1000, Cohesion Biosciences, UK), TIMP-1 (1:1000, Abcam, USA), AKT1 (1:1000, Cohesion Biosciences, UK), VEGFA (1:1000, Abcam, USA), MET (1:1000, Cohesion Biosciences, UK), MMP-9 (1: 1000, Cohesion Biosciences, UK), MTA2 (1:1000, Cohesion Biosciences, UK), and β-actin (1:5000, Cohesion Biosciences, UK) were incubated overnight at 4 °C.

Techniques: Control, Transduction

Journal: Heliyon

Article Title: Mechanism of HIF-1α promoting proliferation, invasion and metastasis of nasopharyngeal carcinoma by regulating MMP-2 in hypoxic microenvironment

doi: 10.1016/j.heliyon.2024.e40760

Figure Lengend Snippet: Confirmation of genes regulated by HIF-1α in HONE1. A. qRT-PCR confirmation of genes regulated by HIF-1α in HONE1, including FAS, BRCA1, TIMP-1, AKT1, VEGFA, MET, MMP-2, MMP-9, MTA2. B. Western blot confirmation of genes regulated by HIF-1α in HONE1, including Fas, BRCA1, TIMP-1, AKT1, VEGFA, MET, MMP-2, MMP-9, MTA2. Full, non-adjusted images of the WB are shown in .

Article Snippet: 10 % skimmed milk powder (solarbio, China) was sealed at room temperature for 2 h. The primary antibodies HIF-1α (1:1000, Abcam, USA), MMP-2 (1:1000, Cohesion Biosciences, UK), Fas (1:1000, Abcam, USA), BRCA1 (1:1000, Cohesion Biosciences, UK), TIMP-1 (1:1000, Abcam, USA), AKT1 (1:1000, Cohesion Biosciences, UK), VEGFA (1:1000, Abcam, USA), MET (1:1000, Cohesion Biosciences, UK), MMP-9 (1: 1000, Cohesion Biosciences, UK), MTA2 (1:1000, Cohesion Biosciences, UK), and β-actin (1:5000, Cohesion Biosciences, UK) were incubated overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot