Journal: Cell reports

Article Title: Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks

doi: 10.1016/j.celrep.2019.08.041

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: KAP-1 (TRIM28) rabbit polyclonal antibody (used for: WB) , Bethyl , Cat# A300-274A; RRID:AB_185559.

Techniques: Recombinant, Transfection, Viability Assay, Imaging, Luciferase, Control, Software, Fluorescence, Microscopy

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs repress EBV lytic genes through the transcriptional corepressor TRIM28. (A and B) BL cells were transfected with empty vector and scrambled siRNA, SZF1 (A) or ZNF557 (B) plasmid and scrambled siRNA, or SZF1 (A) or ZNF557 (B) plasmid and siTRIM28. Cells were exposed to NaB and harvested for determination of relative amounts of transcripts from the EBV latency-to-lytic-cycle switch gene BZLF1 and the early lytic gene BMRF1 by qRT-PCR. Data represent results from two independent experiments with 3 technical replicates each; error bars indicate standard errors of the means (*, p ≤ 0.05). Samples were also harvested for immunoblotting and reacted with indicated antibodies. Representative data from two independent experiments are shown. (C and D) ChIP was performed on BL cells containing doxycycline (dox)-inducible shTRIM28 cassette. Cells were left untreated (open bars) or treated with doxycycline (black bars) for 12 h. DNA precipitated by using anti-H3K9Ac and anti-H3K9Me3 antibodies was analyzed via qPCR using primers spanning predicted SZF1-binding sites in the promoters of lytic genes BZLF1 (C) and BMRF1 (D) and normalized to input. Immunoblot analysis of TRIM28 after doxycycline induction of shTRIM28 was performed to assess knockdown efficiency. Data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: SZF1 and TRIM28 interact directly and colocalize on EBV lytic promoters preferentially in quiescent/latent cells. (A) Coimmunoprecipitation assays using control rabbit IgG or anti-SZF1, anti-ZNF557, or anti-TRIM28 antibody were performed on BL cells that were cotransfected with HA-SZF1 and HA-ZNF557 plasmids. Precipitates were analyzed by probing with anti-TRIM28 or anti-HA antibody to detect pulled-down endogenous TRIM28 or overexpressed, HA-tagged SZF1 or ZNF557, respectively. IP, immunoprecipitation; IB, immunoblotting. (B) Proximity ligation assays using antibodies targeting SZF1 and TRIM28 (left) or ZNF557 and TRIM28 (right) were performed on NaB-treated BL cells. Lytic cells were identified by immunofluorescence staining with anti-ZEBRA antibody. Representative fields from 3 independent experiments are shown. (C) Thirty lytic (ZEBRA+) and 70 refractory (ZEBRA−) nuclei were examined, and the numbers of PLA foci per ZEBRA+ and ZEBRA− nucleus were determined. (D) PLAs using control goat IgG plus control rabbit IgG (−/−), control goat IgG plus rabbit anti-SZF1 antibody (−/SZF1), goat anti-TRIM28 antibody plus control rabbit IgG (TRIM28/−), or goat anti-TRIM28 plus rabbit anti-SZF1 antibodies (TRIM28/SZF1) were performed on BL cells. Fluorescent green foci indicate in situ interactions between TRIM28 and SZF1. Data are representative of results from 3 independent experiments. (E to G) Chromatin precipitated from BL cells using anti-SZF1, anti-TRIM28, or a control rabbit IgG antibody was subjected to qPCR analysis (S, SZF1; T, TRIM28) using primers spanning bioinformatically predicted SZF1-binding sites within BZLF1p (E), BRLF1 (F), and BMRF1p (G) and normalized to input or subjected to a second round of ChIP using anti-TRIM28 or anti-SZF1 antibody (S/T and T/S), respectively, before qPCR analysis. Negligible amounts of DNA were pulled down by using rabbit IgG control antibody, and values were subtracted from experimental results prior to normalization. (H to J) ChIP–re-ChIP was performed, as described above for panels E to G, on untreated and NaB-treated BL cells. Data represent the averages of results from three independent experiments; error bars indicate the standard errors of the means (*, p ≤ 0.05).

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Immunoprecipitation, Western Blot, Ligation, Immunofluorescence, Staining, In Situ, Binding Assay

Journal: Journal of Virology

Article Title: KRAB-ZFP Repressors Enforce Quiescence of Oncogenic Human Herpesviruses

doi: 10.1128/JVI.00298-18

Figure Lengend Snippet: KRAB-ZFPs regulate persistence of KSHV, the other oncogenic human herpesvirus. (A) Levels of SZF1, ZNF557, and STAT3 mRNAs in KSHV+ primary effusion lymphoma (BCBL-1) cells transfected with scrambled siRNA (open bars) or siSTAT3 (black bars) were quantitated by qRT-PCR. BCBL-1 cells were transfected with scrambled siRNA or siSTAT3 and harvested for immunoblotting with indicated antibodies. (B and C) BCBL-1 cells were transfected with empty vector and scrambled siRNA, SZF1 (B) or ZNF557 (C) plasmid and scrambled siRNA, or SZF1 (B) or ZNF557 (C) plasmid and siTRIM28, exposed to VPA, and harvested for determination of the relative amounts of transcripts from the KSHV latency-to-lytic-cycle switch gene ORF50 and the early lytic gene ORF59 by qRT-PCR. All data represent the averages of results from three independent experiments; error bars indicate standard errors of the means (*, p ≤ 0.05). (D and E) BCBL-1 cells were transfected with combinations of empty vector, HA-tagged KRAB-ZFP plasmids, scrambled siRNA, and siTRIM28, treated with VPA, and harvested for immunoblotting with indicated antibodies. Data are representative of results from two independent experiments. (F) Model of oncogenic human herpesvirus persistence depicting that STAT3 transcriptionally activates the KRAB-ZFPs SZF1 and ZNF557, which localize to EBV and KSHV genomes. Bound SZF1 functions directly through TRIM28 to repress lytic genes despite the presence of lytic triggers, while bound ZNF557 functions in a TRIM28-independent manner (?), thereby maintaining latency and promoting virus persistence.

Article Snippet: Rabbit anti-STAT3 (catalog number sc-482) and anti-SZF1 (catalog number sc-100263) antibodies were purchased from Santa Cruz Biotechnology; rabbit anti-ZNF557 antibodies (catalog numbers sc-130005 and 20500-1-AP) were obtained from Santa Cruz Biotechnology and Proteintech, respectively; anti-TRIM28 antibodies (catalog numbers A300-274A, A303-838A, and ab22553) were purchased from Bethyl Laboratories and Abcam; mouse anti-β-actin (catalog number AC-15) and anti-HA (catalog number H3663) antibodies were obtained from Sigma; and anti-H3 (ab1791), anti-acetylated H3 (catalog number ab47915), and anti-trimethylated lysine 9 H3 (catalog number ab8898) antibodies were purchased from Abcam.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Plasmid Preparation

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: Representative immunohistochemical staining of TRIM28, ×400

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Immunohistochemical staining, Staining

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: Associations between the expression of TRIM28 and clinicopathologic characteristics of the 136 cases of ESCC patients

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Expressing

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: Associations between the expression of TRIM28, clinicopathological parameters and 5-year overall survival rate of the 136 cases of ESCC patients

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Expressing

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: Relative expression of TRIM28 protein by Western blot between paired ESCC tissues and NEE tissues (Cells in ESCC can express TRIM28 while there was no expression of TRIM28 in cells of NEE)

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Expressing, Western Blot

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: The distribution of TRIM28 protein in ESCC and NEE tissues by immunofluorescence stainings, ×400. (TRIM28 protein was stained with green fluorescence(→), which was expressed in the nucleus in ESCC cells and nuclei were counterstained with DAPI (A); Analysis results of the fluorescence intensity (B), P = 0.000) (One-way ANOVA)

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Pathology Oncology Research

Article Title: Expression and Significance of TRIM 28 in Squamous Carcinoma of Esophagus

doi: 10.1007/s12253-018-0558-6

Figure Lengend Snippet: TRIM28 protein expression during cancer progression by IHC analysis

Article Snippet: After washing 5 min in PBS for three times, the sections were incubated overnight at 4 °C with rabbit polyclonal anti-TRIM28 antibody (GTX102226; dilution, 1:200; GeneTex, USA) and followed by incubation with reagent I of HRP-labeled goat anti-rabbit IgG antibody (SP-9001, Zhongshan Goldenbridge Biol\Technology Co., Ltd., Beijing, China) for 30 min at 37 °C.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation

doi: 10.1038/srep39049

Figure Lengend Snippet: ( A , B ) Co-immunoprecipitation of interacting proteins with REST. Co-immunoprecipitation was performed by mixing V5 antibody-conjugated beads with cell lysates of HEK293FT cells transfected with pcDNA_V5 or pcDNA_REST-V5 vectors. mRNA processing/splicing associated target proteins including ALYREF and HnRNP Q ( A ) and transcription-related target proteins including HDAC5, NPM1, NCL, PARP1, and TRIM28 ( B ) were confirmed by co-immunoprecipitation coupled with western blotting. Arrows indicate the target proteins. Full-length blots are included in the . ( C , D ) Co-localization of REST with interacting proteins such as ALYREF, HnRNP Q ( C ), HDAC5, NPM1, NCL, PARP1, and TRIM28 ( D ). HEK293FT cells transfected with REST-V5 expressing vector were fixed after 24 h, followed by probing with the indicated target specific antibody and V5 antibody. Images analyzed at magnification of 400x. Scale bar indicates 10 μm.

Article Snippet: Antibodies for immunoprecipitation and western blot were from the companies as follows; rabbit anti-REST antibodies (Millipore, Abcam), mouse anti-V5 antibody (Sigma), mouse ant-ALY antibody (Santa Cruz), mouse anti-HnRNP Q (Santa Cruz), anti-HDAC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling) and rabbit and mouse anti-TRIM28 antibodies (Santa Cruz, Abcam).

Techniques: Immunoprecipitation, Transfection, Western Blot, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation

doi: 10.1038/srep39049

Figure Lengend Snippet: ( A ) The overlap between differentially expressed transcripts following knockdown of REST and REST-interacting proteins is depicted as Venn diagrams. Blue and green circles indicate transcripts regulated by REST and TRIM28, respectively. ( B ) Gene Ontology (GO) analysis using gene sets upregulated by REST and TRIM28 knockdown. ( C ) Network analysis of gene set upregulated by REST and TRIM28 knockdown. Edges were drawn based on the STRING databases. The node color indicates the clustering coefficency. Fisher’s test was used for statistical analysis.

Article Snippet: Antibodies for immunoprecipitation and western blot were from the companies as follows; rabbit anti-REST antibodies (Millipore, Abcam), mouse anti-V5 antibody (Sigma), mouse ant-ALY antibody (Santa Cruz), mouse anti-HnRNP Q (Santa Cruz), anti-HDAC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling) and rabbit and mouse anti-TRIM28 antibodies (Santa Cruz, Abcam).

Techniques:

Journal: Scientific Reports

Article Title: Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation

doi: 10.1038/srep39049

Figure Lengend Snippet: ( A ) Physical interaction between REST and TRIM28 in the mouse brain lysates. ( B ) SH-SY5Y cells transfected with an indicated siRNA. Levels of REST, TRIM28 and actin were assessed using western blot. Full-length blots are included in the . ( C ) mRNA levels of CTNND2 in SH-SY5Y cells transfected with the indicated siRNA are indicated in the histogram. Three independent experiments were performed, and the t-test was used for statistical analysis. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Antibodies for immunoprecipitation and western blot were from the companies as follows; rabbit anti-REST antibodies (Millipore, Abcam), mouse anti-V5 antibody (Sigma), mouse ant-ALY antibody (Santa Cruz), mouse anti-HnRNP Q (Santa Cruz), anti-HDAC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling) and rabbit and mouse anti-TRIM28 antibodies (Santa Cruz, Abcam).

Techniques: Transfection, Western Blot

Journal: Scientific Reports

Article Title: Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation

doi: 10.1038/srep39049

Figure Lengend Snippet: ( A ) Primary neurons were obtained from mouse embryo and maintained in the culture plate. The primary neurons were fixed on days 0, 2 and 4, followed by probing with Phalloidin. Scale bar indicates 50 μm. ( B – D ) mRNA levels of REST ( B ), TRIM28 ( C ) and protein levels of REST, TRIM28 ( D ) were accessed in the primary neurons on days 0, 2 and 4. ( E ) mRNA level of CTNND2 accessed in the primary neurons on days 0, 2 and 4. Three independent experiments were performed to analyze alteration of genes at mRNA level, and the t-test was used for statistical analysis. (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: Antibodies for immunoprecipitation and western blot were from the companies as follows; rabbit anti-REST antibodies (Millipore, Abcam), mouse anti-V5 antibody (Sigma), mouse ant-ALY antibody (Santa Cruz), mouse anti-HnRNP Q (Santa Cruz), anti-HDAC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling) and rabbit and mouse anti-TRIM28 antibodies (Santa Cruz, Abcam).

Techniques:

Journal: Scientific Reports

Article Title: Interactomic analysis of REST/NRSF and implications of its functional links with the transcription suppressor TRIM28 during neuronal differentiation

doi: 10.1038/srep39049

Figure Lengend Snippet: ( A ) Representative images of differentiated CAD cells transfected with pmCherry-N1 and siRNAs. Scale bar indicates 30 μm. ( B ) The longest neurite length normalized to soma diameter in siRNA-mediated knockdown groups was normalized to that in the control group (n = 50 for control siRNA, 35 for REST siRNA, 33 for TRIM28 siRNA, 41 for REST siRNA + TRIM28 siRNA). ( C ) Representative images of cultured primary cortical neurons transfected with pmCherry-N1 and siRNAs. Scale bar indicates 20 μm. ( D ) The longest neurite length normalized to soma diameter in siRNA-mediated knockdown groups was normalized to that in the control group (n = 27 for control siRNA, 22 for REST siRNA, 19 for TRIM28 siRNA). The t-test was used for statistical analysis. (* P < 0.05; ** P < 0.01; *** P < 0.001) ( E ) REST and TRIM28 might repress the expression levels of CTNND2 in neural progenitor cells. As the neural progenitor cells differentiate, levels of REST and TRIM28 decrease, which may induce expression of CTNND2 as a result of the loss of transcriptional suppression by REST and TRIM28.

Article Snippet: Antibodies for immunoprecipitation and western blot were from the companies as follows; rabbit anti-REST antibodies (Millipore, Abcam), mouse anti-V5 antibody (Sigma), mouse ant-ALY antibody (Santa Cruz), mouse anti-HnRNP Q (Santa Cruz), anti-HDAC5 (Santa Cruz), rabbit anti-B23 antibody (Santa Cruz), rabbit anti-C23 antibody (Santa Cruz), rabbit anti-PARP1 antibody (Cell signaling) and rabbit and mouse anti-TRIM28 antibodies (Santa Cruz, Abcam).

Techniques: Transfection, Cell Culture, Expressing