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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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

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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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: DNA plasmids DNA plasmids used in this study for ectopic expression included: Fuw-Flag-Mbd3 (Addgene 52371), FUW-Flag-Mbd3 delta1-70 (Addgene 52372), FUW-2XFlag-ΔCCR-Mbd3 (Deletion of 220-279), FUW-Gatad2a, FUW-Gatad2b, pCAG-HA-Gatad2a-CCR (140-174 of Gatad2a), pCAG-HA-(MBD)Mbd3 (1-170 of Mbd3), pCAG-HA-GFP, FUW-Gatad2a-mutatnt ((K30R, K485R) SUMOylation-sites).

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

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: The relative expression of MBD3 in pan-cancer and COAD. ( A , B ) Different types of tumors compared with normal tissues in TCGA and GTEx databases. ( C , D ) Relative expression of MBD3 in colon cancer and colon cells. ( E ) Relative expression of MBD3 in single cells of COAD (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Expressing

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: Clinical indicators of MBD3 in colon cancer. ( A ) Overall survival analyses of MBD3 in COAD. ( B ) Risk score of MBD3 in COAD. ( C , D ) Nomogram and calibration curves for prediction of one-, two-, and three-year overall survival rates of patients with COAD with high expression of MBD3. ( E ) Prognostic values of MBD3 expression by univariate analysis in COAD. ( F ) Prognostic values of MBD3 expression by multivariate analysis in COAD.

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Expressing

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: The immune mutational landscape, MSI and MATH of MBD3 in pan-cancer. ( A ) Immune mutational landscape of MBD3 in pan-cancer. ( B , C ) MSI and MATH of MBD3 in pan-cancer.

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques:

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: Function enrichment of MBD3 in single cells and DEGs. ( A , B ) Relationship between MBD3 expression and different functional states in tumors explored by the CancerSEA tool. * p < 0.05, ** p < 0.01. ( C – E ) Volcano plot of logFc > 1.5 and the top ten related differentially expressed genes of MBD3 from the TCGA database. ( F – H ) KEGG, GO and GSEA analysis of MBD3 and their co-expression genes.

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Expressing, Functional Assay

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: Representative images of MBD3 expression in COAD tissues and their matched paracancerous tissues. Original magnifications 20× and 40× (inset panels). ( A – C ) were representative images.

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Expressing

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: Cytological experiments regarding MBD3 (ns, ( D ) * p ≥ 0.05; ( E ) * p < 0.05; ( H , L ) * p < 0.01; ( J , N ) * p < 0.001). ( A , D ) The relative expression of MBD3 in COAD cell lines. ( B , C , F ) Plasmid transfection efficiency of MBD3 in COAD cell lines. ( G ) Cell migration assay of MBD3 in SW620, HCT116, SW480 and CaCO2. ( H ) Cell invasion assay of MBD3 in SW620, HCT116, SW480 and CaCO2. ( I – L ) show enhanced ability of MBD3 in COAD.

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Migration Assay, Invasion Assay

Journal: Cancers

Article Title: MBD3 as a Potential Biomarker for Colon Cancer: Implications for Epithelial-Mesenchymal Transition (EMT) Pathways

doi: 10.3390/cancers15123185

Figure Lengend Snippet: Effect of MBD3 on colon cancer proliferation and tumor formation experiment in nude mice. ( A – D ) Proliferative capacity of MBD3 in COAD. ( E – H ) Clone formation assay of MBD3 in COAD. ( I – L ) Tumor formation in nude mice (* p < 0.05).

Article Snippet: After deparaffinization, rehydration and microwave antigen retrieval, the slides were incubated with MBD3 (Proteintech Cat No. 14258-1-AP) antibody at 1:800 dilution at 4 °C overnight.

Techniques: Tube Formation Assay

Journal: American Journal of Translational Research

Article Title: Methyl-CpG-binding domain 3 (Mbd3) is an important regulator for apoptosis in mouse embryonic stem cells

doi:

Figure Lengend Snippet: Sequences of primers used for qPCR

Article Snippet: The Mbd3 overexpression plasmids, FUW-Mbd3 (#52356) and control FUW-M2rtTA (#20342), were purchased from Addgene.

Techniques:

Journal: American Journal of Translational Research

Article Title: Methyl-CpG-binding domain 3 (Mbd3) is an important regulator for apoptosis in mouse embryonic stem cells

doi:

Figure Lengend Snippet: Mbd3 overexpression induced cellular apoptosis. A. Overexpression of Mbd3 RNA levels was achieved by transfection with the FUW-Mbd3 plasmid in fifth-passage mES. The FUW-M2rtTA plasmid and the PKCi group were used as controls. B. Mbd3 protein expression was evaluated by Western blot in fifth-passage mES (upper panel). Quantitative density analysis showed that Mbd3 overexpression resulted in its increased protein levels compared with the PKCi control (lower panel). C. Mbd3 overexpression significantly reduced the total number of fifth-passage mES. D. CCK8 assay revealed that Mbd3 overexpression decreased mES cell viability. E. Apoptosis was evaluated by Annexin V staining in fifth-passage mES. The thick arrow indicates cell nuclei (blue), and the thin arrow indicates apoptotic cells (red). Scale bar =100 μm (left panel). The apoptosis rate was significantly increased with Mbd3 overexpression (right panel). F. Bax, Bcl-2, and β-actin protein levels were evaluated by Western blot in fifth-passage mES (left panel). Quantitative density analysis showed similar levels of Bax in all three groups (middle panel) and significantly reduced Bcl-2 levels with Mbd3 overexpression (right panel). G. Mbd3 overexpression increased the expression of Bax, Bim, Trail, Fasl, and caspase 3 RNA, but reduced the expression of Bcl-2 RNA, in fifth-passage mES. The data are represented as mean ± SD (n=3). *P<0.05, **P<0.01.

Article Snippet: The Mbd3 overexpression plasmids, FUW-Mbd3 (#52356) and control FUW-M2rtTA (#20342), were purchased from Addgene.

Techniques: Over Expression, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, CCK-8 Assay, Staining

Journal: American Journal of Translational Research

Article Title: Methyl-CpG-binding domain 3 (Mbd3) is an important regulator for apoptosis in mouse embryonic stem cells

doi:

Figure Lengend Snippet: Mbd3 knockdown partially reversed the cellular apoptosis induced by PKCi removal. A. Mbd3 RNA expression levels were increased in third-passage mES when PKCi was removed for 48 h from the culture medium, but they were only partially decreased with Mbd3 knockdown. B. Mbd3 protein levels were assessed by Western blot in third-passage mES (upper panel). Quantitative density analysis showed a significant increase in Mbd3 upon PKCi removal, and a partial reduction was observed with Mbd3 knockdown. C. The total number of third-passage mES was dramatically decreased upon PKCi removal and increased with Mbd3 knockdown, but it was still lower than that of the PKCi group. D. Cell viablility was improved by Mbd3 knockdown. E. Cellular apoptosis was assessed by Annexin V staining in third-passage mES. The thick arrow indicates cell nuclei (blue), and the thin arrow indicates apoptotic cells (red). Scale bar =100 μm (left panel). The apoptosis rate was significantly increased upon PKCi removal, but it was partially reduced with Mbd3 knockdown (right panel). F. Bax, Bcl-2, and β-actin levels were evaluated by Western blot in third-passage mES with Mbd3 knockdown (left panel). Quantitative density analysis showed increased levels of Bax after PKCi removal, but only partially reduced levels were observed with Mbd3 knockdown (middle panel). Bcl-2 expression was reduced upon PKCi removal, and similar levels were observed with Mbd3 knockdown (right panel). G. PKCi removal resulted in increased expression levels of Bax, Bim, Trail, Fasl, and caspase 3 RNA, with reduced Bcl-2 levels, in third-passage mES. Mbd3 knockdown resulted in partially reduced Bax, Bim, Trail, and caspase 3 RNA levels, unchanged Fasl levels, and increased Bcl-2 levels. The data are represented as mean ± SD (n=3). The letters a, b, c indicate significant differences among the groups (P<0.05).

Article Snippet: The Mbd3 overexpression plasmids, FUW-Mbd3 (#52356) and control FUW-M2rtTA (#20342), were purchased from Addgene.

Techniques: Knockdown, RNA Expression, Western Blot, Staining, Expressing