coding region of gfp  (GenScript corporation)

Journal: Science advances

Article Title: Lnc956 -TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis.

doi: 10.1126/sciadv.adf6277

Figure Lengend Snippet: Fig. 5. Lnc956-TRIM28-HSP90B1 associate with each other on replication forks. (A) Mass spectrometry (MS) analyses identified TRIM28 and HSP90B1 as candidates to reside on replication forks and interact with Lnc956. (B) In vivo RNA pulldown validated the interaction of Lnc956 with HSP90B1 and TRIM28. The interaction was enhanced by HU (top) or aphidicolin (Aph) treatment (bottom). Pulldown assay using sense probe was included as negative control (NC). (C and D) Reciprocal immunoprecipitation (IP) validated the physical interaction of HSP90B1 with TRIM28. The interaction was enhanced by HU (C) or aphidicolin (D) treatment and required the mediation of RNA species. IgG, immunoglobulin G. (E) Immunoblotting (IB) of iPOND samples validated the localization of HSP90B1 and TRIM28 on replication forks of ESCs under normal culture condition. Cells were chased with 10 μM thymidine chase for different times. (F) The accumulation of HSP90B1 and TRIM28 on replication forks was dynamically regulated by HU (left) or aphidicolin treatment (right). The relative protein levels were normalized by input (B to D) or by histone H3 (E and F), and the levels in samples marked with box were set as 1. In (B) to (F), all experiments were repeated three times with similar results.

Article Snippet: The Lnc956 and Trim28 coding regions (CDS) were inserted into pTOMO–internal ribosomal entry site (IRES)–enhanced green fluorescent protein (EGFP) lentiviral expression vector (Addgene, #26291).

Techniques: Mass Spectrometry, In Vivo, Negative Control, Immunoprecipitation, Western Blot

Journal: Science advances

Article Title: Lnc956 -TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis.

doi: 10.1126/sciadv.adf6277

Figure Lengend Snippet: Fig. 6. Lnc956-TRIM28-HSP90B1 form an interdependent complex on stalled replication forks. (A) iPOND showed that Lnc956 KO impaired the recruitment of HSP90B1 and TRIM28 on stalled replication forks at all time points of HU treatment. Reexpression of Lnc956 (KO-rescue) rescued the defects. (B and C) Reciprocal IP showed that Lnc956 KO weakened the TRIM28-HSP90B1 interaction under replication stress. The defect was rescued by reexpression of Lnc956. (D) TRIM28 KD (KD-1) reduced the accumulation of HSP90B1 on stalled replication forks. (E) iROND were performed using same numbers of cells. qRT-PCR analysis of iROND samples showed that Lnc956 levels on replication forks were increased by fork stalling in TRIM28-proficient ESCs, whereas fork-resided Lnc956 was reduced upon fork stalling in TRIM28 KD ESCs. (F) TRIM28 KD compromised the association of HSP90B1 with Lnc956 as revealed by in vivo RNA pulldown. (G to I) Similarly, HSP90B1 KD (KD-1) decreased the accumulation of TRIM28 (G) and Lnc956 (H) on stalled replication forks and impaired the interaction of TRIM28 with Lnc956 (I). In vivo RNA pulldown using sense probe was set as negative control (NC) in (F) and (I). The protein levels were normalized by histone H3 in (A), (D), and (G) and by input in (B), (C), (F), and (I). The relative protein levels in samples marked with box were set as 1. All experiments were repeated three times with consistent results. Data were shown as mean ± SEM, two-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The Lnc956 and Trim28 coding regions (CDS) were inserted into pTOMO–internal ribosomal entry site (IRES)–enhanced green fluorescent protein (EGFP) lentiviral expression vector (Addgene, #26291).

Techniques: Quantitative RT-PCR, In Vivo, Negative Control, Two Tailed Test

Journal: Science advances

Article Title: Lnc956 -TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis.

doi: 10.1126/sciadv.adf6277

Figure Lengend Snippet: Fig. 7. Lnc956-TRIM28-HSP90B1 complex physically interacts with MCM hexamer via TRIM28. (A) MCM7 coimmunoprecipitated with TRIM28 and HSP90B1. The interaction was enhanced by HU treatment and did not rely on RNA species. (B and C) Reciprocally, TRIM28 (B) and HSP90B1 (C) could immunoprecipitate with MCM7 but not PSF1 or CDC45. The interaction was also stimulated by HU treatment and did not rely on RNAs. (D) KD of TRIM28 (top) or HSP90B1 (bottom) in Lnc956 KO mESC using two independent Dox-inducible shRNAs. (E and F) KD of TRIM28 in Lnc956 KO ESCs impaired the association of HSP90B1 with MCM7, as revealed by reciprocal immu- noprecipitation. (G and H) However, KD of HSP90B1 in Lnc956 KO ESCs had no influence on the interaction between TRIM28 and MCM7, as shown by reciprocal immu- noprecipitation. The relative protein levels were normalized by input, and the levels in samples marked with box were set as 1. All experiments were repeated three times with consistent results.

Article Snippet: The Lnc956 and Trim28 coding regions (CDS) were inserted into pTOMO–internal ribosomal entry site (IRES)–enhanced green fluorescent protein (EGFP) lentiviral expression vector (Addgene, #26291).

Techniques:

Journal: Science advances

Article Title: Lnc956 -TRIM28-HSP90B1 complex on replication forks promotes CMG helicase retention to ensure stem cell genomic stability and embryogenesis.

doi: 10.1126/sciadv.adf6277

Figure Lengend Snippet: Fig. 8. HSP90B1 stabilizes CMG helicase under replication stress. (A and B) iPOND (A) and chromatin purification (B) analyses showed that HSP90B1 mutation (R448A) did not affect the recruitment of HSP90B1 and TRIM28 to replication forks. However, R448A mutation, similar to HSP90B1 KD, induced the premature dissociation of CMG helicase from stalled replication forks. (C) HSP90B1 R448A mutation robustly increased the K48 and K63 ubiquitylation of MCM7 under the HU treatment condition. (D and E) HSP90B1 R448A mutant ESCs were treated with HU to induce K48 and K63 ubiquitylation of MCM7. Under this condition, blocking P97 activity by CB-5083 or NMS-873 preserved CMG retention on stalled replication forks, as shown by iPOND (D) and chromatin purification analyses (E). (F) HSP90B1 R448A mutation impaired the stalled fork restart. At least 200 fibers from three independent experiments were analyzed. (G) The working model of Lnc956-TRIM28-HSP90B1 complex on replication forks. The relative protein levels were normalized by histone H3 in (A), (B), (D), and (E). The levels in samples marked with box were set as 1. All experiments were repeated three times with similar results. Data were shown as mean ± SEM, two-tailed Student’s t test. ***P < 0.001.

Article Snippet: The Lnc956 and Trim28 coding regions (CDS) were inserted into pTOMO–internal ribosomal entry site (IRES)–enhanced green fluorescent protein (EGFP) lentiviral expression vector (Addgene, #26291).

Techniques: Purification, Mutagenesis, Blocking Assay, Activity Assay, Two Tailed Test

Journal: Human Molecular Genetics

Article Title: Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1 -associated neurodevelopmental disorders

doi: 10.1093/hmg/ddac200

Figure Lengend Snippet: Dominant negative activity of de novo DEAF1 variants. (A) Immunofluorescent images of 293 T stable cells expressing GFP, WT-DEAF1, or the indicated DEAF1 heritable (p.R226W) or de novo variants (p.P174_G222del, p.Q264P) using anti-FLAG antibodies. (B) Western blots were performed on proteins isolated from 293 t stable cells using anti-FLAG (DEAF1) and anti-tubulin antibodies. (C) RT-qPCR was performed using RNA isolated from HEK293T Control and HEK293T stable cells to determine changes in the expression of the indicated DEAF1-regulated genes. Each bar represents mean +/− SEM (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared to DEAF1 using one-way analysis of variance followed by Dunnett’s multiple comparisons test.

Article Snippet: FLAG-tagged WT or indicated DEAF1 variant coding sequences were used to replace the GFP coding region in peGIP lentiviral plasmid (Addgene #26777) ( 38 ).

Techniques: Dominant Negative Mutation, Activity Assay, Expressing, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: Human Molecular Genetics

Article Title: Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1 -associated neurodevelopmental disorders

doi: 10.1093/hmg/ddac200

Figure Lengend Snippet: Effects of DEAF1 WT and p.Q264P on DEAF1-regulated gene expression in cells lacking DEAF1. (A) HEK293T DEAF1-KO (CRISPR/Cas9) cells were transduced with AAV particles that express GFP, DEAF1, or DEAF1-p.Q264P. RT-qPCR was performed to determine changes in the expression of the indicated DEAF1-regulated genes. Each bar represents mean +/− SEM (N = 4). *P < 0.05, **P < 0.01, ***P < 0.001 compared to GFP-transduced Control using one-way analysis of variance followed by Fisher’s least square comparisons test. #P < 0.01 compared to GFP-transduced DEAF1-KO using one-way analysis of variance followed by Fisher’s least square comparisons test. (B) Models of WT-DEAF1, DEAF1 KO and dominant negative actions of identified DEAF1 variants on DEAF-target gene regulation.

Article Snippet: FLAG-tagged WT or indicated DEAF1 variant coding sequences were used to replace the GFP coding region in peGIP lentiviral plasmid (Addgene #26777) ( 38 ).

Techniques: Gene Expression, CRISPR, Transduction, Quantitative RT-PCR, Expressing, Control, Dominant Negative Mutation