Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) Dual luciferase assay of 293T cells co-transfected with the luciferase constructs containing the SETDB1 3′-UTR_WT or SETDB1 3′-UTR_Mut as well as miR-29s mimics or Scr NC. ( B ) SETDB1 protein levels was assessed in PC14, A549, and BEAS-2B cells. ( C ) SETDB1 mRNA levels and ( D ) SETDB1 protein levels after transfection of miR-29s mimics or Scr NC into A549 and PC14 cells. ( E ) SETDB1 mRNA levels and ( F ) SETDB1 protein levels after transfection of miR-29s inhibitors or Scr NC into BEAS-2B cells. * P <0.05; ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Luciferase, Transfection, Construct

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: miR-29s mRNA expression after transfection of pEGFP-TP53 vector or pEGFP-N1 control vector into ( A ) A549 cells and ( B ) PC14 cells. ( C ) SETDB1 protein level and ( D ) SETDB1 mRNA expression after transfection of pEGFP-TP53 vector or pEGFP-N1 control vector into A549 and PC14 cells. ( E ) miR-29s mRNA expression, ( F ) SETDB1 mRNA expression and ( G ) SETDB1 protein level after transfection of siTP53 or Scr NC into BEAS-2B cells. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Expressing, Transfection, Plasmid Preparation, Control

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) TP53 mRNA levels and ( B ) TP53 protein levels ( C , D ) miR-29s levels after transfection of siSETDB1 scrambled oligonucleotides as the negative control into A549 and PC14 cells. ( E ) TP53 mRNA levels and ( F ) TP53 protein levels and ( G ) miR-29s levels after transfection of pcDNA3.1-SETDB1 vector or pcDNA3.1 control vector into BEAS-2B cells. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Transfection, Negative Control, Plasmid Preparation, Control

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) SETDB1 mRNA levels and ( B ) TP53 mRNA levels in primary NSCLC samples and their paired adjacent normal samples. ( C ) SETDB1 and TP53 mRNA levels were inversely correlated in primary NSCLC samples. ( D – F ) Pearson correlation analysis between miR-29s and SETDB1 mRNA levels, as well as between miR-29s and TP53 mRNA levels in primary NSCLC samples. *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques:

Journal: Bioscience Reports

Article Title: A regulatory circuitry comprising TP53, miR-29 family, and SETDB1 in non-small cell lung cancer

doi: 10.1042/BSR20180678

Figure Lengend Snippet: ( A ) TP53 and Suv39h1 mRNA levels after transfection of miR-29s mimics or Scr NC into A549 and PC14 cells. ( B ) TP53 and Suv39h1 mRNA levels after transfection of miR-29s inhibitors or Scr NC into BEAS-2B cells. Histone H3K9 methylation status in ( C ) NSCLC cells (A549 and PC14) with overexpressed miR-29s and ( D ) BEAS-2B cells with downexpressed miR-29s were examined by Western blot using antibodies specific for H3K9me2 and H3K9me3 (di- and trimethylation of H3K9, respectively); expression levels of histone H3 protein were used as an internal control. ( E ) Schematic illustration of the proposed TP53/ miR-29s /SETDB1 regulatory circuitry in NSCLC. ** P <0.01; *** P <0.001

Article Snippet: The siRNAs specifically targetting SETDB1 (siSETDB1) and TP53 (siTP53) were obtained commercially (GenePharma, Shanghai, China).

Techniques: Transfection, Methylation, Western Blot, Expressing, Control

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: SETDB1 expression decreases with chronological aging. Immunofluorescence (IF) analysis of normal human fibroblast from healthy female (ages: 20, 40 and 60) and male (age: 20, 40 and 60) donors. IF was performed with (A) anti-CSB (red) and (B) anti-SETDB1 (green). Cell nuclei were stained with DAPI. Representative images are maximum intensity z-projection of all the slices from z-stacks. Quantification of mean fluorescence intensity of z-stacks of cells from different sex and aged groups, or different aged groups to measure staining intensity. For each cell line from an individual, a total of 19 and 13 cells were analyzed for CSB and SETDB1, respectively. Error bars represents SEM. (C) Brain lysates of young, middle and old mice were probed for the indicated proteins on western blot. Blot density was measured and normalized to GAPDH (H3K9me3 was normalized to H3). Scale bar, 30 μm. Error bars represent SD. NS, not significant; *, <0.05; **, <0.01.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: H3K9me3 recovery by SETDB1 reduces PAR production by oxidative stress. Tet-inducible SETDB1 (A) or SUV39H1 (B) expressing CSB-deficient (CSB−) cells were treated with 10 or 50 ng/ml of doxycycline for 72 h. Whole cell lysates were probed for the indicated proteins on western blot. Sample loading quantities were normalized with either total H3 or GAPDH. Tet-inducible SETDB1 (C) or SUV39H1 (E) cells were treated with DMSO or 50 ng doxycycline for 72 h and treated with 2.5 mM H2O2 for 30 min before ADPr-ChAP sample preparation. Chromatin samples prepared for ADPr-ChAP were probed for nuclear PAR analysis. (D and F) ADPr-ChAP were performed from chromatin preparations from C and E and analyzed for real-time qPCR on indicated genomic locus. Error bars represent SD. *, <0.05.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Western Blot, Sample Prep

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: SETDB1-induction restores mitochondrial homeostasis in CS fibroblasts. (A) Doxycycline-induced HMTase expression was probed on western blot (CSB+; CSB-corrected cells, SUV; SUV39H1-inducible CSB-deficient cells, SET; SETDB1-inducible CSB-deficient cells). (B) H3K9me3-ChIP and ADPr-ChAP sequencing reads of SETDB1-inducible CSB-deficient (CSB- rtTA SETDB1) cells were aligned on TSS. (C and D) Merged dataset profiles of aligned sequencing reads from (B). Seahorse extracellular flux analysis representative trace (E) and quantification of basal respiration (F), proton leak (G), maximal respiration (H), complex IV function (I), spare capacity (J), ATP-linked respiration (K) and non-mitochondrial respiration (L) in CSB-corrected (CSB+) and SUV39H1/SETDB1-inducible CSB-deficient cells. (n = 5; data are represented as mean ± SEM) *P< 0.05, **P< 0.01, ***P< 0.001 One-way ANOVA.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Western Blot, Sequencing

Journal: Nucleic Acids Research

Article Title: Cockayne syndrome group B deficiency reduces H3K9me3 chromatin remodeler SETDB1 and exacerbates cellular aging

doi: 10.1093/nar/gkz568

Figure Lengend Snippet: Schematic diagram of CSB-SETDB1 axis. In CS, as well as chronological aging, decreased CSB causes decreased SETDB1 expression, which could lead to reduced H3K9me3 on TSS regions, triggering accumulation of PAR and mitochondrial abnormalities.

Article Snippet: Dox-inducible GFP-tagged lentiviral SUV39H1 or SETDB1 constructs were designed and ordered from VectorBuilder Inc. (Santa Clara, CA, USA).

Techniques: Expressing

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: Tiam1 could interacted with SETDB1directly by GST- pull down and Cross-linking assays. A Schematic representation of the domain structure of Tiam1. PCER; C685;C751 and C1199 were four truncations constructed containing different domains. B Interaction sites was verified by GST-pull down and westernblot assays. Proteins pulled down by agarose beads with GST tag further were detected by westernblot, (a) purified Tiam1-PCER protein labelled with GST was co-incubated with purified SETDB1 and detected by westernblot after elution, as shown in figure.a, Tiam1-PCER could be detected but not STEDB1, indicating the Tiam1-PCER fragment has no binding sties with SETDB1 (b,c,d) purified Tiam1-C685 protein was obtained as indicated, as shown in above figures, Tiam1-C685, C751 and C1199 could be detected as well as STEDB1, indicating the these fragments have binding sties with SETDB1. C Cross-linking assay confirmed Tiam1could interacted with SETDB1

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Construct, Purification, Incubation, Binding Assay

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 promoted cell proliferation both in vivo and vitro in HCC. a The expression of SETDB1 was detected by westernblot after transfecting with overexpression and knockdown virus. b Overexpression of SETDB1 could promote cellsproliferation ability was detected by CCK8 and Plate clony formation assays. ** P <0.01. c knockdown of SETDB1 could inhibit cellsproliferation ability was detected by CCK8 and Plate clony formation assays. ** P <0.01. d SETDB1 could promote HCC cell proliferation in vitro by xenografts experiments. ** P <0.01

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: In Vivo, Expressing, Over Expression, In Vitro

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 promoted cell metastasis both in vivo and vitro in HCC. a and b Transwell and Boyden assays indicate SETDB1 could enhance cell migration and invasion. The invasive cells were stained and counted under microscope at 24–30 h after reseeding. Original magnification, × 400. ** P <0.01, as compared LV-SET with WT and LV-con groups, or LV-SET with WT and LV-shcon groups. c SETDB1 significantly promoted HCC tumorigenicity in vivo as demonstrated by an orthotopic tumor implantation experiment in nude mice. d SETDB1 increased lung metastasis in an orthotopic tumor implantation model in nude mice. Hematoxylin and eosin staining confirming the formation of HCC tumor foci in the lungs. e SETDB1 modulates EMT-related genes expression

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: In Vivo, Migration, Staining, Microscopy, Tumor Implantation, Expressing

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: SETDB1 expression is associated with Tiam1expression in HCC cancer. a The expression of SETDB1 and Tiam1 in HCC biopsy samples and control normal samples detected by qRT-PCR. b SETDB1correlates positively with Tiam1 in HCC tissue samples. c The expression of SETDB1 in HCC tissues and adjacent normal tissue were detected by IHC

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: BMC Cancer

Article Title: Histone methyltransferase SETDB1 promotes cells proliferation and migration by interacting withTiam1 in hepatocellular carcinoma

doi: 10.1186/s12885-018-4464-9

Figure Lengend Snippet: Tiam1 is a functional target of SETDB1. a The expression of Tiam1 was identified by westernblot analysis. b Knockdown of Tiam1 could offset the effect of SETDB1 on cell migration ability by transwell assay. c Knockdown of Tiam1 could decrease the number of colonies caused by SETDB1

Article Snippet: The purified protein SETDB1 was acquired with TNT® Quick Coupled Transcription/Translation Systems (Promega,USA).

Techniques: Functional Assay, Expressing, Migration, Transwell Assay

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Eleven high-confidence ^a non-synonymous variants identified by whole exome ^b and targeted deep sequencing ^c

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Sequencing, Mutagenesis

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: SETDB1 mutations identified in 78 MPMs from 69 patients and previously reported

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Mutagenesis

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Two types of deletion mutations from MPM patients, shown by an Integrative Genomics Viewer (IGV). A. 17 bp deletion (677_693del17) and B. In-frame deletion (3747_3749del) of SETDB1 were identified from different MPM patients.

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques:

Journal: Oncotarget

Article Title: Whole exome and targeted deep sequencing identify genome-wide allelic loss and frequent SETDB1 mutations in malignant pleural mesotheliomas

doi: 10.18632/oncotarget.7032

Figure Lengend Snippet: Nine SETDB1 somatic mutations identified in this study and reported previously were mapped in domains of SETDB1 . Four of five truncating mutations were located at the N-terminal (5′) regions before Tudor (Tud) domains. All three missense and one deletion mutation were found in the SET domain. Tud: Tudor; MBD: methyl-CpG-binding domain; pS: pre-SET; PS: post-SET [ – ].

Article Snippet: We designed another targeted panel of SETDB1 covering all 21 coding exons spanning 6.05 kb (99.76% coverage) (CureSeq Inc, Brisbane, CA).

Techniques: Mutagenesis, Binding Assay