mbd3 sirna  (Santa Cruz Biotechnology)

Journal: Cell stem cell

Article Title: Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1016/j.stem.2018.07.004

Figure Lengend Snippet: A. MEFs harboring TetO-OKSM and M2rtTA cassettes were transfected with siRNA targeting different canonical NuRD components (indicated in the illustration), 2 and 4 days after reprogramming initiation following DOX administration. Reprogramming was then evaluated by AP staining at day 8. B. Reprograming efficiency following siRNA treatments was evaluated using AP staining, after 8 days of reprogramming (n=3, two-sided Student’s t-test p values are indicated). C. Cell growth curves of MEFs treated with siRNA for the indicated NuRD components (two-sided Student’s t-test p values are indicated). D. Representative images of cells treated with siRNA targeting Mbd3 or Gatad2a and exposed to BrdU in order to evaluate proliferation. E. Quantitative evaluation of BrdU incorporation test (n=8, two-sided Student’s ttest p values are indicated Student’s t-test). F. Viability and apoptosis induction were measured using FACS following Annexin-PI staining. G. Reprogramming efficiency following siRNA treatments targeting different NuRD components, at different time points. KD was performed at two distinct cycles: the early one (Regimen 1, marked in black) started one day prior to DOX induction, and the second one (Regimen 2, marked in grey) started one day post-DOX induction. H. iPSC reprogramming efficiency following different siRNA treatments. was evaluated at day 8. (n=3 per each condition, two-sided Student’s t-test p values are indicated).

Article Snippet: siRNA targeting Mouse-Mbd3 , Invitrogen , MSS-237238.

Techniques: Transfection, Staining, BrdU Incorporation Assay

Journal: Cell stem cell

Article Title: Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1016/j.stem.2018.07.004

Figure Lengend Snippet: A. Mass spectrometry analysis of NuRD complex components binding efficiency to Mbd3 in Gatad2a-KO and its isogenic Gatad2a-WT line, in MEF cells during reprogramming by OKSM. Only putative NuRD components are presented. B. Flag-Mbd3 CoIP in Gatad2a-WT and Gatad2a-KO cells. Experiments were conducted both in MEF cells and ESs. C. Cells during reprogramming were treated with siRNA targeting Gatad2a, and pellets collected after four days. CoIP of Chd4 shows that siGatad2a prevents Mbd3 binding to Gatad2a but also to Chd4. D. Gatad2a-KO MEF with overexpression of Gatad2a (transgenic recovery; abbreviated as Gatad2a-Tg) or Mock were subjected to Chd4-CoIP. E. The coiled coil region of Mbd3 is highly conserved between different organisms, and different proteins in the MBD family. The highlighted amino acids are crucial for Gatad2a binding to Mbd2 or Mbd3. F. A scheme of Flag tagged Mbd3, and mutant forms of Mbd3: lacking the Coiled coil region (ΔCCR-Mbd3) or the methyl-binding domain (ΔMBD-Mbd3). G. WT-Mbd3 and both mutants were over-expressed in 293T cells and were subjected to Flag-Mbd3 CoIP to examine their protein interactions. H-I. Mbd3fl/- secondary MEF, harboring ΔPE-Oct4-GFP reporter, were transfected with two different forms of Flag tagged WT-Mbd3 and ΔCCR-Mbd3. The cells were then subjected to reprogramming. (two-sided Student’s t-test, n=3. p Value<0.0001).

Article Snippet: siRNA targeting Mouse-Mbd3 , Invitrogen , MSS-237238.

Techniques: Mass Spectrometry, Binding Assay, Over Expression, Transgenic Assay, Mutagenesis, Transfection

Journal: Cell stem cell

Article Title: Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1016/j.stem.2018.07.004

Figure Lengend Snippet: A. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were subjected to different differentiation protocols, and cells from 5 distinct states (naïve ESC, EpiLC, EBs, MEF, 4-day OKSM Reprogramming) were subsequently subjected to CoIP with anti-Flag-Mbd3. Lysates were then analyzed by western blot, and reacted with different antibodies against different NuRD components, pluripotency factors, and other epigenetic proteins. B. Rosa26-M2rtTA Col1a:TetO-2XFlag-Mbd3 ES cells were either maintained in ground state naïve conditions or in priming condition. Lysates were subjected to Co-IP with anti-Flag-Mbd3 and examined by Western blot. Oct4 protein binding to Mbd3 can be detected only in the primed pluripotent state. C. Mbd3 expression in ES cells treated with growth media containing different small molecules, after 72 hours of treatment. D. PKCi Go6983 effect on Mbd3 level is seen after approximately 48 hours, in different concentration. E. Western blot showing Mbd3 protein levels in V6.5 ESCs expanded in different conditions. F. Western blot analysis for different NuRD components in ESC cells with and without Go6983. G. Mbd3 levels in MEFs following treatment with Go6983. H. RT-PCR analysis for Mbd3 transcript abundance following Go6983 treatment. I. Go6983 causes mild depletion in Mbd3 in MEFs following 3 days of OKSM expression. J. Gatad2a+/+ WT MEFs carrying ΔPE -Oct4-GFP reporter and constitutive mCherry markers (secondary system i) were subjected to iPSC reprogramming protocols in in Fig. 2A and iPSC efficiency was quantified at day 8. In the last two conditions included in the panel, the MEFs were pre-treated with control and Ubc9 shRNA following with Neomycin selection (Cheloufi et al., 2015), and then subjected to DOX mediated iPSC reprogramming. K. WT EpiSCs reversion efficiency to naïve ESCs in different conditions. Anova test P values are indicated. L. Isogenic WT and Gatad2a KO ESCs were expanded on feeder free plates in N2B27 LIF only or LIF/PKCi conditions. Phase images and Oct4-GFP signal maintenance are shown after 8 passages (P8).

Article Snippet: siRNA targeting Mouse-Mbd3 , Invitrogen , MSS-237238.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Protein Binding, Expressing, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, shRNA, Selection

Journal: Cell stem cell

Article Title: Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1016/j.stem.2018.07.004

Figure Lengend Snippet: A. ES cells treated with naïve ground state condition were treated with shRNA targeting Ubc9 or Scramble. Cells were lysed and subsequent CoIP of Chd4 shows a decrease in Gatad2a binding. B. Western blot analysis for protein abundance before and after knockdown of Ubc9 in ESCs. C. 2-d08 specific small molecule inhibitor for SUMOylation was applied, and co-IP experiments of Mbd3 shows a decrease in Gatad2a and Chd4 binding. D. Cells induced in naïve ground state 2i/LIF conditions and subsequent shRNA targeting either for Ubc9 or scramble negative control. The cells were lysed and fractioned – Cytoplasm, Nucleoplasm and Chromatin fractions, proteins were analyzed by western blot. E. As in D, with immunoblot for SUMO2/3 on the same exact gel series. F. Schematic representation of Gatad2a known domains and functionally validated SUMOylation sites. G. Western blot analysis for Gatad2a protein expression following lentiviral infection in Gatad2a-/- MEFs. H. Representative FACS images for reprogramming efficiency following Mock, Gatad2a-WT and Gatad2a-MUT (K30R, K485R) lentiviral infection of Gatad2a-/- secondary MEFs. I. Quantitative summary of results for experiment elucidated in H.

Article Snippet: siRNA targeting Mouse-Mbd3 , Invitrogen , MSS-237238.

Techniques: shRNA, Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Negative Control, Expressing, Infection

Journal: Cell stem cell

Article Title: Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naïve Pluripotency

doi: 10.1016/j.stem.2018.07.004

Figure Lengend Snippet: List of RT-PCR primers used in this study.

Article Snippet: siRNA targeting Mouse-Mbd3 , Invitrogen , MSS-237238.

Techniques: Recombinant, Protease Inhibitor, Sample Prep, Methylation, Knock-In, Negative Control, Reverse Transcription Polymerase Chain Reaction, Software

Journal: Nature Communications

Article Title: Netrin-1 regulates somatic cell reprogramming and pluripotency maintenance

doi: 10.1038/ncomms8398

Figure Lengend Snippet: ( a ) Representative FACS profile of thy1 High and thy1 Neg cells 3–5 days post OSKM infection. Cells were sorted and plated back in reprogramming media for 6–9 additional days before AP activity staining. ( b ) Representative FACS profile of apoptosis in thy1 Neg cells following Ntn1 and/or DCC depletion. Apoptotic cells percentages are indicated. ( c ) Quantification of thy1 Neg apoptotic cells following Ntn1 and/or DCC depletion at days 3 and 5. The apoptotic cell percentage in sh-scrambled thy1 Neg MEFs is set at 100%. Data are the mean±s.d. ( n =3). Student's t -test, * P <0.05 or ** P <0.01 or *** P <0.001; NS, non-significant. ( d ) Ntn1 and DCC expression levels in thy1 Neg cells. Cells were sorted at days 3 and 6 post OSKM retroviral infection. Quantitative reverse transcription–PCR (Q-RT–PCR) data are expressed relative to MEF as the mean±s.d. ( n =3). ( e ) Scheme depicting Netrin-1 promoter constructs: part A, B and A+B . pGL3-control vector with SV40 promoter and enhancer driving luciferase expression is used as ‘control vector'. ( f ) Dox-inducible MEFs were transfected with Netrin-1 promoter luciferase constructs and treated with or without dox for 48 h. Data are normalized to Renilla activity and expressed as the mean±s.d. ( n =2). Student's t -test, P <0.05. ( g ) Regulation of Netrin-1 promoter activity by single reprogramming factors. HEK293T cells were transfected with Netrin-1 promoter part A+B and reprogramming factors. Data are the mean±s.d. ( n =3). Student's t -test, P <0.05. ( h ) Mbd3 is recruited to Ntn1 promoter upon reprogramming. ChIP-seq resource was employed to assess Ntn1 promoter occupancy in mouse ES cells and in MEF following OSKM induction. Data are extracted from refs , . Mbd3 is recruited on chromosome 11 (position 68209776-68210746 NCBI36/mm8) in the vicinity of Oct4- (chr11 68210308-68210332), Sox2- (chr11 68210051-68210500) and Klf4- (chr11 68210297-68210303) binding sites. ( i ) Netrin-1 transcriptional silencing is rescued by Mbd3 depletion. MEFs were transfected with Mbd3 siRNA 24 h before OSKM infection and cells harvested 36 h post OSKM infection. Data are the mean±s.d. ( n =3). Student's t -test, * P <0.05 or ** P <0.01. ( j ) OSKM repressive effect on Ntn1 promoter activity is mediated by Mbd3. HEK293T cells were transfected with Mbd3 siRNA before Ntn1 promoter luciferase construct and reprogramming factors. Luciferase activity was assessed 48 h post transfection and normalized to Renilla activity. Data are the mean±s.d. ( n =2). Student's t -test, * P <0.05. ( k ) Netrin-1 silencing is mediated by Mta1 and Chd4. MEFs were infected with lentiviral particles targeting the NuRD complex members Mta1, Mta2 and Chd4 and reprogramming induced 48 h later. Q-RTPCR depicts Ntn1 , Mta1 , Mta2 and Chd4 expression levels at reprogramming day 2. Data are the mean±s.d. ( n =3). Student's t -test, * P <0.05 or ** P <0.01.

Article Snippet: Human Mbd3 stealth siRNA were employed (Life Technologies, hss147580 and hss147581).

Techniques: Infection, Activity Assay, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Construct, Plasmid Preparation, Luciferase, Transfection, ChIP-sequencing, Binding Assay

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: FBI-1 interacts directly with MBD3. ( A ) Co-IP and western blot analysis. HEK293T-REX-control and stable HEK293T-REX-FLAG-FBI-1 cell nuclear extracts were immunoprecipitated with anti-FLAG M2 agarose beads, and the precipitates were analysed by western blot using the indicated antibodies. ( B ) Reverse co-IP of ( A ). The same nuclear extracts were immunoprecipitated with an antibody against MBD3, and the precipitates were analysed by western blot using the anti-FLAG antibody. ( C ) GST-fusion protein pull-down assays. Schematic diagram of the domains of FBI-1 tested. Recombinant GST protein, GST-POZFBI-1 and GST-ZFDBDFBI-1 were incubated with in vitro synthesized [ 35 S]-methionine-labelled MBD3, precipitated, resolved by 10% SDS-PAGE and analysed by autoradiography.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Recombinant, Incubation, In Vitro, Synthesized, SDS Page, Autoradiography

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 enhances transcriptional repression of the CDKN1A gene by FBI-1. ( A ) Structures of the two CDKN1A- luc fusion reporter constructs tested. ( B and C ) Transient transcription assays in HEK293 cells. Cells were transfected with each luciferase reporter construct and the expression vectors for MBD3 and FBI-1. The average of three independent assays is shown; bars represent standard deviations. ( D ) Western blot analysis. HEK293 cells were transfected with MBD3 and/or FBI-1 expression vector. Cell lysates were analysed for endogenous p21 expression. Ectopic FBI-1 and MBD3 expression potently repress endogenous CDKN1A expression. ( E ) Transient transcription assays in HEK293 cells. Knock-down of FBI-1 or/and MBD3 derepressed reporter gene expression from the p21WAF/CDKN1A -Luc (−131 bp) construct. Luciferase activities were normalized to protein concentration. The average of three independent experiments is shown. Bars represent standard deviations. ( F ) Western blot analysis. HEK293 cells were transfected with MBD3 siRNA and/or FBI-1 siRNA. Cell lysates were analysed for endogenous p21 expression. Knock-down of endogenous FBI-1 and/or MBD3 expression increases endogenous p21 expression. Negative control, scrambled siRNA.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Construct, Transfection, Luciferase, Expressing, Western Blot, Plasmid Preparation, Protein Concentration, Negative Control

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 enhances transcriptional repression of pG5-5x(FRE)-Luc by FBI-1. FBI-1 binds to both methylated and unmethylated FRE elements of CDKN1A , and MBD3 binding to the FRE probes is increased by ectopic FBI-1. ( A ) Transient transcription assays in HEK293 cells and Drosophila SL2 cells. Cells were transfected with the reporter construct, mammalian or Drosophila (pPac) expression vector of FBI-1 and/or MBD3. All data shown are the average of three independent assays; bars represent standard deviations. ( B ) EMSA. 32 P-labelled FRE and methylated FRE probes were incubated with recombinant GST-ZF-FBI-1 protein (500 ng), separated by 4% non-denaturing polyacrylamide gel electrophoresis, and the dried gel was exposed to radiographic film. ( C ) Oligonucleotide pull-down assay. Nuclear extracts from HEK293 control or HEK293 cells transfected with the FLAG-FBI-1 expression vector were incubated with biotinylated double stranded oligonucleotides corresponding to the FRE and methylated FRE. The precipitated complexes were analysed by western blotting using the anti-FLAG and anti-MBD3 antibodies. GAPDH was used as a control.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Methylation, Binding Assay, Transfection, Construct, Expressing, Plasmid Preparation, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Pull Down Assay, Western Blot

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 binding to the FRE element requires FBI-1 in vivo . ( A ) Methylation of the FBI-1 responsive reporter, pG5-5x(FRE)-Luc, with the methylase SssI. Plasmid methylation was tested by digestion with methylated DNA-specific endonucleases BstUI and HpaII. M, DNA size marker. Lanes 1–3, unmethylated plasmid. Lanes 4–6, methylated plasmid. ( B ) ChIP assays of FBI-1 and MBD3 binding on the proximal promoter of pG5-5x(FRE)-Luc and the methylated plasmids transfected into HEK293 cells. ChIP primer sets were designed to amplify the region flanking the proximal FRE elements. Arrows indicate the primer-binding sites. ( C ) ChIP assays of FBI-1 and MBD3 binding in Drosophila SL2 cells lacking mammalian transcription factors. MBD3 binding requires FBI-1. ( D ) Structure of the human CDKN1A gene promoter. Arrows at the promoter regions indicate the locations of ChIP PCR primer-binding sites. ( E ) qChIP assays of MBD3 binding to the endogenous CDKN1A promoter under ectopic expression of FBI-1 and/or MBD3, or knock-down of MBD3 in HEK293 cells. MBD3 binding is increased by ectopic FBI-1 and/or MBD3 ( F ) qChIP assays of FBI-1 in the presence of ectopic FBI-1 and/or MBD3. ( G–J ) ChIP-reChIP assays of FBI-1 and MBD3 binding at the proximal CDKN1A promoter by ectopic expression or knock-down of either FBI-1 or MBD3. The negative controls were IgG and the 3′-UTR.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Binding Assay, In Vivo, Methylation, Plasmid Preparation, Marker, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 increases the protein interaction between FBI-1 and co-repressor BCoR. The MBD3–FBI-1 interaction and BCoR recruitment decreases histone acetylation and histone H3K4-Me3 but increases histone H3K9-Me3. ( A ) Mammalian two-hybrid assay of molecular interaction between co-repressor and FBI-1. HEK293 cells were transiently transfected with the plasmids indicated (200 ng of reporter plasmid, GAL4 fused FBI-1-ZF, VP-16 fused proteins and MBD3 expression vector). All data represent the average of three independent assays and bars represent standard deviations. ( B ) Co-IP and western blotting. HEK293 cells were transfected with the expression vectors, cell lysates were immunoprecipitated and analysed by western blotting using the indicated antibodies. ( C ) Structure of the human CDKN1A gene promoter. Arrows indicate the locations of ChIP PCR primer-binding sites. ( D ) qChIP assays of co-repressor switching (NCoR, BCoR) at the proximal CDKN1A promoter by knock-down of MBD3 expression. The binding of FBI-1 and MBD3 is also shown. The negative control was IgG. ( E ) qChIP assays of co-repressor, FBI-1 and MBD3 binding at the 3′-UTR of CDKN1A , a qChIP assay control DNA region. The negative control was IgG. ( F ) Semi-quantitative ChIP assay of histone modifications at the proximal promoter of endogenous CDKN1A gene by ectopic FBI-1 and/or MBD3 expression. Histone marks indicating active transcription (acetylated H3 and H4, H3K4-Me3) and repressed transcription (H3K9-Me3) were tested. ( G ) qChIP analysis of histone modifications (H3K4-Me3; H3K9-Me3) at the proximal promoter of the endogenous CDKN1A gene by ectopic expression or knock-down of MBD3 and/or MBD3 expression. N.C., negative control scrambled siRNA. ( H ) qChIP analysis of histone modifications (H3K4-Me3; H3K9-Me3) on the endogenous CDKN1A gene by knock-down of MBD3 or BCoR. N.C., negative control scrambled siRNA. ( I ). qChIP analysis of BCoR and MBD3 binding to the endogenous CDKN1A gene after knock-down of MBD3 or BCoR expression. ( J ) RT-qPCR analyses of knock-down efficiency of MBD3 or BCoR mRNA by siRNA.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Two Hybrid Assay, Transfection, Plasmid Preparation, Expressing, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation, Binding Assay, Negative Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: The CDKN1A gene is repressed by HDAC and DNA methylation; FBI-1 and MBD3 are important in DNA methylation. ( A ) Effect of the HDAC inhibitor, TSA (200 nM), and DNA methylation inhibitor, 5-aza-2′-deoxycytidine (1 µM), on the transcription of pGL2- CDKN1A -Luc (−131 bp) repressed by FBI-1 and/or MBD3. ( B ) qChIP assays of acetylated H3 and H4, H3K4-Me3 and H3K9-Me3 around the exogenous proximal CDKN1A promoter introduced by transfection, before and after treatment with TSA and 5-aza-2′-deoxy-cytidine. ( C ) Structure of the human CDKN1A gene promoter. Arrows at the promoter region indicate the locations of ChIP PCR primer sets. ( D–F ) Me-DIP assay of DNA methylation at the proximal promoter of the endogenous CDKN1A gene after ectopic expression or knock-down of FBI-1 and/or MBD3. Me-PC, methylation positive control (a positive signal is obtained for methylation) (Diagenode); Me-NC, methylation negative control (no signal is obtained for 0% methylation); GAPDH, GAPDH promoter (no signal is expected as this region is not methylated); Alpha X1, X-linked alpha-satellites (a positive signal is expected as it is a methylated region). (F) qRT-PCR showed that FBI-1 and MBD3 expression are knocked down efficiently. ( G and H ) Bisulfite DNA sequencing assays. The percentage of methylated cytosines in the CDKN1A promoter under various assay conditions is shown. Cells were transfected with expression vectors or siRNA containing knocked down FBI-1 and/or MBD3, and DNA was prepared for bisulfite sequencing. Individual PCR products were cloned, and 20 clones were sequenced. Filled circles on the proximal promoter sequence indicate the CpG sites that can be methylated.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: DNA Methylation Assay, Transfection, Expressing, Methylation, Positive Control, Negative Control, Quantitative RT-PCR, DNA Sequencing, Methylation Sequencing, Clone Assay, Sequencing

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: FBI-1 and BCoR interact with the MBD3-Mi-2/NuRD-HDAC complex, DNMT1, DNMT3b and HP1. ( A ) Co-IP and western blot analysis of FBI-1, MBD3, the Mi-2/NuRD-HDAC complex, DNMTs and HP1. HEK293 cells were transfected with the FBI-1 and/or MBD3 expression vector and immunoprecipitated using an anti-FBI-1 antibody. The immunoprecipitates were analysed for the presence of FBI-1, MBD3, the Mi-2/NuRD-HDAC complex, DNMTs and HP1. GAPDH was used as the loading control. ( B ) Co-IP and western blot analysis of FBI-1, BCoR, MBD3, the Mi-2/NuRD-HDAC complex, DNMTs and HP1. HEK293 cells were transfected with the BCoR expression vector and immunoprecipitated using an anti-BCoR antibody. The immunoprecipitates were analysed for the presence of FBI-1, MBD3, the Mi-2/NuRD-HDAC complex, DNMTs and HP1. GAPDH was used as the loading control.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 and BCoR play critical role in the recruitment of Mi-2/NuRD-HDAC complex and subsequent binding of DNMTs and HP1. ( A ) Structure of human CDKN1A gene promoter. Arrows indicate the locations of qChIP PCR primer binding sites. ( B ) qChIP assays of Mi-2/NURD-HDAC complex (as tested for diagnostic subunit MTA2) binding at the endogenous CDKN1A promoter of the cells transfected with FBI-1 or/and MBD3. ( C ) qChIP assays of the Mi-2/NuRD-HDAC complex binding at the endogenous CDKN1A promoter after knockdown of MBD3 or BCoR. Knockdown of MBD3 or BCoR expression decrease Mi-2/NuRD-HDAC complex binding. ( D ) qChIP assays of DNMT1 binding in HEK293 cells. HEK293 cells were transfected with ectopic expression vectors or knockdown siRNA of MBD3 or/and FBI-1. Chromatin was fixed and DNMT binding was analyzed by qChIP. ( E ) qChIP assays of the DNMT1 binding at the endogenous CDKN1A promoter after knockdown of MBD3 or BCoR. Knockdown of MBD3 or BCoR expression potently decrease DNMT recruitment. ( F ) qChIP assays of HP1 binding to the endogenous CDKN1A promoter in HEK29A cells, in the presence of ectopic MBD3, FBI-1 or siRNA against MBD3 or BCoR. ( G ) qChIP assays of the HP1 binding at the endogenous CDKN1A promoter after knockdown of MBD3 or BCoR. Knockdown of MBD3 or BCoR expression decrease HP1 recruitment. ( H ) qChIP assays of DNMT1 or 3b binding. HEK293 cells were transfected with HP1 siRNA and analyzed for DNMTs binding.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Binding Assay, Diagnostic Assay, Transfection, Expressing

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: MBD3 and FBI-1 repress the transcription of CDKN1A by histone modification and DNA methylation in primary human HDFn cells. ( A and B ) RT-qPCR and western blot analysis of p21 expression in HDFn cells transfected with FBI-1 and/or MBD3 expression vectors. Ectopic FBI-1 and/or MBD3 decrease endogenous p21 expression. ( C and D ) RT-qPCR and western blot analysis of p21 expression in HDFn cells transfected with siRNA designed to knock down endogenous FBI-1 and/or MBD3 expression. Knock-down of FBI-1 and/or MBD3 increases endogenous p21 mRNA expression. ( E ) Structure of the human CDKN1A gene promoter. Arrows indicate the locations of the ChIP PCR primer-binding sites. ( F ) qChIP analysis of histone modifications at the proximal CDKN1A promoter caused by FBI-1 and/or MBD3 expression. Histone marks indicating active transcription (acetylated H3 and H4, H3K4-Me3) and repressed transcription (H3K9-Me3) were tested. The negative control was IgG. ( G ) qChIP analysis of histone modifications at the proximal promoter of the endogenous CDKN1A gene by knock-down of endogenous FBI-1 and/or MBD3 expression. The negative control was IgG. ( H and I ) Me-DIP assay of DNA methylation at the proximal CDKN1A promoter after ectopic expression or knock-down of FBI-1 and/or MBD3. Me-PC, methylation positive control; Me-NC, methylation negative control; GAPDH, GAPDH promoter; Alpha X1, X-linked alpha-satellites.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: Modification, DNA Methylation Assay, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Binding Assay, Negative Control, Methylation, Positive Control

Journal: Nucleic Acids Research

Article Title: The proto-oncoprotein FBI-1 interacts with MBD3 to recruit the Mi-2/NuRD-HDAC complex and BCoR and to silence p21WAF/CDKN1A by DNA methylation

doi: 10.1093/nar/gkt359

Figure Lengend Snippet: Hypothetical model of epigenetic silencing of the CDKN1A gene by FBI-1, MBD3 and BCoR, and DNA methylation by DNMT1/3b. ( A ) In normal cells with no or low FBI-1, transcription of CDKN1A is mainly regulated by Sp1 family proteins acting on GC-boxes of the proximal promoter, and cells express basal levels of p21. PCAF/p300, co-activator with HAT activity. Histones are acetylated and rich in H3K4-Me3. Few CpG are methylated. ( B ) With a high level of FBI-1, FBI-1 binds to the FRE (GC-box #3) critical in transcription initiation and activation, and interacts with a co-repressor (SMRT/NCoR) to repress transcription by deacetylation of histones H3 and H4, demethylation of H3K4-Me3 and methylation of H3K9. In this case, SMRT/NCoR competes with BCoR to interact with FBI-1. ( C ) In the presence of MBD3 and FBI-1, MBD3 binds to the zinc-finger domain of FBI-1 and recruits the Mi-2/NuRD-HDAC complex and directs FBI-1 to interact primarily with BCoR co-repressor. MBD3 and BCoR play critical roles in the recruitment of the Mi-2/NuRD-HDAC complex. Eventually, DNMT1/3b and HP1 proteins associated with the NuRD complex are recruited to methylate the promoter DNA, which forms a heterochromatin structure in the region.

Article Snippet: Also, primary human dermal fibroblast neonatal (HDFn) cells were transfected with various combinations of pcDNA3.0-FLAG-FBI-1 (5 μg), pcDNA3.1-MBD3 (5 μg), FBI-1 siRNA (100 pmoles) and/or MBD3 siRNA (100 pmoles) using the Neon Kit of the Neon Transfection System (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions ( Supplementary Table 1 ).

Techniques: DNA Methylation Assay, Activity Assay, Methylation, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 1. Immunostaining for mBD3 and mBD4 in infected cornea at 1 day p.i. For mBD3 (A and B), staining was similar in infected cornea of B6 and BALB/c mice. For mBD4 (C and D), the corneal stroma of B6 mice was stained more intensely than in BALB/c mice, while no difference was seen in the corneal epithelium between the two groups. Controls, in which the primary Ab was replaced by donkey IgG, were negative for immuno- staining for mBD3 or mBD4 and appeared similar to SYTOX Green Nu- clear staining (E and F). Magnification, 100. Images shown are repre- sentative of two repeated experiments each with three mice per group.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: Immunostaining, Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 2. Immunostaining for mBD3 and mBD4 in infected cornea at 5 days p.i. For mBD3 (A and B), the corneal epithelium of BALB/c mice was stained more intensely than in B6 mice, while no difference was shown in the corneal stroma of the two groups. For mBD4 (C and D), both corneal epithe- lium and stroma of B6 mice was stained more intensely than in BALB/c mice. Controls, in which the primary Ab was replaced by donkey IgG, were negative for immunostaining for mBD3 or mBD4 and appeared similar to SYTOX Green Nuclear staining (E and F). Magnification, 100. Images shown are representative of two repeated experiments each with three mice per group.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: Immunostaining, Infection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 3. In vivo knockdown studies of mBD3 and mBD4. For mBD3 siRNA treatment, clinical scores (A) indicated statistically signifi- cant differences at 3 and 5 days p.i., compared with controls. Photographs taken with a slit lamp of infected eyes at 5 days p.i. displayed more opacity and a worsened disease response when comparing control (B) vs mBD3 siRNA (C) treatment. For mBD4 siRNA treatment, clinical scores (D) and similar photographs indicated no differences between control (E) and mBD4 silenced (F) mice after infection. RT-PCR (G–J) confirmed the efficacy and specificity of each silencing. Magnification, 6. Data are the means SEM and represent two individual experiments each with five animals/group/time/assay. , p 0.05; , p 0.01; , p 0.001 by Mann-Whitney U or Student’s t test; ns, not significant.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: In Vivo, Knockdown, Infection, Control, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 4. In vivo silencing of mBD2, mBD3, and both defensins. Clinical scores (A) and photographs taken with a slit lamp of P. aerugi- nosa-infected eyes at 5 days p.i. displayed an enhanced disease level and more opacity when comparing control (B) vs siRNA treatment for mBD2 (C), mBD3 (D), or both defensins (E). Bacterial plate counts (F) and PMN recruitment as detected by MPO activity (G) are shown in each group. Magnification, 6. Data are the means SEM and represent two indi- vidual experiments each with five animals/group/time/assay. , p 0.05; , p 0.01; , p 0.001 by Mann-Whitney U or Student’s t test; ns, not significant.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: In Vivo, Infection, Control, Activity Assay, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 5. Efficacy and specificity of in vivo siRNA treatment. RT- PCR confirmed that knockdown of mBD2 (A), mBD3 (C), and both de- fensins (E and F) vs control treatment was effective at 5 days p.i., and that mBD3 mRNA levels in mBD2 siRNA-treated mice (B) as well as mBD2 mRNA levels in mBD3 siRNA-treated mice (D) were unchanged at that time, with no differences detected in normal, uninfected tissue for either defensin. Data are the means SEM and represent two individual exper- iments each with five animals/group/time/assay. , p 0.05; , p 0.01; , p 0.001 by Student’s t test; ns, not significant. FIGURE 6. Protein expression levels after P. aeruginosa infection. Pro- tein levels of MIP-2 (A), TNF- (B), and IL-1 (C) were significantly up-regulated in siRNA mBD2, mBD3, and both defensins vs scrambled control-treated mice at 5 days p.i. Silencing both defensins also elevated protein levels of MIP-2, TNF-, and IL-1 at 5 days p.i., when compared with silencing either defensin alone. Data are the means SEM and rep- resent two individual experiments each with 5 animals/group/time. , p 0.05; , p 0.01; , p 0.001 by Student’s t test; ns, not significant.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Knockdown, Control, Expressing, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Beta-defensins 2 and 3 together promote resistance to Pseudomonas aeruginosa keratitis.

doi: 10.4049/jimmunol.0902140

Figure Lengend Snippet: FIGURE 7. mBD2 and mBD3 staining in BALB/c-infected stroma. mBD2-positive staining was found in fibroblasts (A), M (C), and PMN (E). PMN also stained positively for mBD3 (G). Controls (B, D, F, H), in which the primary Abs were replaced by the same host IgG, were negative for staining and appeared similar to SYTOX Green Nuclear staining (not shown). Magnification, 500. Images shown are representative of two repeated experiments each with three mice per group.

Article Snippet: For the studies described herein, siRNA for mBD2, mBD3, mBD4, and both mBD2 and mBD3 (composed of an equal mixture of mBD2 and mBD3 siRNA at the same concentration) or appropriate scrambled controls for each (Santa Cruz Biotechnology) were injected subconjunctivally (5 l per mouse at a concentration of 8 M) into the left eye of BALB/c mice (n 5/group/ time) 1 day before infection and then topically applied onto the infected corneas (5 l/mouse/time at a concentration of 4 M, once on the day of infection and twice on both 1 and 3 days p.i.).

Techniques: Staining, Infection