pcr amplified e2f4 cdna fragments  (Cell Signaling Technology Inc)

Journal: Nature communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression.

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: Fig. 3 | Deletion of Trim33 promotes E2f4 interactions with chromatin and with the Recql helicase. a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33- WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33- KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a The top significantly represented transcription factors targeting Trim33-deregulated genes in p19/Nras cells; data were analyzed using Enrichr . b Cycloheximide chase assays in p19/Nras Trim33-WT and Trim33-KO cells; n = 3. Right panel shows the mean intensity (±SD) for three independent experiments with linear regression analysis. c Ubiquitin pulldown assays in HeLa cells transfected with vectors encoding E2f4, his-Ub, and wildtype (WT) or catalytically inactive (CA) Trim33; n = 3. d Immunoprecipitation assays with E2f4 antibodies in p19/Nras Trim33-WT cells treated with MG132 or DMSO; n = 3. e PLA assays in p19/Nras Trim33-WT with antibodies to Trim33 and E2f4. 50 cells per group were analyzed; n = 3. Scale bar = 2 μm. f Schematic of E2f4 domain structure and the variants used in the following experiments. g Immunoprecipitation analysis following transient transfection of the indicated E2f4 variants in HeLa cells; n = 2. h GST-pulldown assays with the indicated GST-E2f4 fusion proteins and U2OS whole cell lysates; n = 3. i DNA fiber assays in p19/Nras Trim33-WT and Trim33-KO cells transfected with siRNA against E2f4 or a non-targeting control. 100 fibers were analyzed per condition; n = 2. j DNA fiber assays in p19/Nras Trim33-WT and Trim33-KO cells, expressing indicated E2f4 variants, after release from a 4 h HU treatment. 140 fibers were counted per condition; n = 2. e , i , j Significance was determined by Kruskal–Wallis test with Dunn’s multiple comparisons. e , i , j Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Transfection, Immunoprecipitation, Control, Expressing

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Genome browser tracks of E2f4 ChIP-seq in p19/Nras Trim33-WT and Trim33-KO cells; n = 2. b Quantification of ChIP-seq tag coverage at E2f4 peaks in p19/Nras Trim33-WT and Trim33-KO cells. c Relationship between changes (log2FC) in E2f4 binding (normalized E2f4 ChIP-seq tags) and mRNA expression (log2FC) of the encoded gene for p19/Nras Trim33-KO vs Trim33-WT cells. Each dot is a bin of 5 genes sorted by the change in mRNA levels. Trendlines are derived from linear regression analysis. d LC-MS/MS analysis of E2f4 interactome in p19/Nras Trim33-WT and Trim33-KO cells. Known interaction partners are highlighted in blue. e PLA analysis of Recql-E2f4 association in p19/Nras Trim33-WT and Trim33-KO cells stably expressing HA-tagged E2f4 or vector Ctrl; from left, n = 49,49,50,50 cells; scale bar = 2 μm; n = 3 independent experiments. f Immunoprecipitation of endogenous E2f4 from benzonase-treated lysates of p19/Nras Trim33-WT and Trim33-KO cells using a mouse anti-E2f4 antibody. Quantification of three biological replicates shows mean intensity and SD. g GST-pulldown assay with the indicated GST-E2f4 fusion proteins and shTrim33 U2OS whole cell lysates; n = 3. h PLA analysis of Recql-E2f4 association in p19/Nras Trim33-KO cells stably expressing the indicated HA-tagged E2f4 variants; from left, n = 100,100,97,100 cells; scale bar = 2 μm; n = 2 experiments. e , h Significance was determined by Kruskal–Wallis test with Dunn’s multiple comparisons. e , h Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: ChIP-sequencing, Binding Assay, Expressing, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Stable Transfection, Plasmid Preparation, Immunoprecipitation, GST Pulldown Assay

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunoprecipitation from p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql; n = 3. b Representative genome browser tracks of Cut&Run analysis of HA-Recql binding in p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql. Lower two tracks—E2f4 ChIP-seq. c Sequencing tag coverage for Recql Cut&Run signal from the experiment shown in b ) at all TSS and at E2f4 sites. d Immunoprecipitation analysis with Recql antibodies from formaldehyde-crosslinked p19/Nras Trim33-WT and Trim33-KO cells, transfected with E2f4-targeting or a control siRNA. e Cut&Run assays with Recql antibodies in p19/Nras Trim33-WT and Trim33-KO cells followed by qPCR analysis at the TSS sites of indicated genes. Mean values of percent input for three technical replicates and standard deviation are plotted. f Representative genome browser tracks of the Cut&Run assays with Recql antibodies in Trim33-KO p19/Nras cells expressing the indicated E2f4 variants. g Cut&Run sequencing tag coverage at all TSS for the experiment shown in f ). h DNA fiber assays in Trim33-KO cells, expressing siRNA against Recql or control siRNA, untreated or released from a 4 h HU treatment; n = 2. 100 fibers were counted per sample and the data were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison. Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. i Immunoblotting analysis of Recql expression in Trim33-KO cells, used in h ). Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunoprecipitation, Expressing, Binding Assay, ChIP-sequencing, Sequencing, Transfection, Control, Standard Deviation, Comparison, Western Blot

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunoprecipitation of endogenous Recql from formaldehyde-crosslinked p19/Nras Trim33-WT cells, untreated or treated with HU for 10 or 120 min; n = 3. b PLA assays with antibodies to biotinylated EdU-labeled nascent DNA and Recql in Trim33-WT and Trim33-KO cells after release from a 4 h HU treatment; from left, n = 112,62,143,113 cells; scale bar = 5 µm; n = 3 experiments. Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. c Representative genome browser tracks of HA-Recql Cut&Run in p19/Nras Trim33-WT and Trim33-KO cells, untreated or treated with HU for 4 h. d Sequencing tag coverage for Recql Cut&Run experiment shown in c ). e Ubiquitin pulldown assay in HeLa cells, transfected with vectors for His-Ub, HA-tagged E2f4, and Trim33-WT or Trim33-CA; n = 2. f Immunoprecipitation of endogenous E2f4 from benzonase-treated lysates of control or HU-treated (2 h) p19/Nras Trim33-WT cells. Quantification of four independent experiments is shown as mean and S.D. of signal intensity g PLA assays with Trim33 and E2f4 antibodies in untreated and HU-treated (2 h) p19/Nras Trim33-WT cells. Significance was determined by a two-tailed t -test. From left, n = 98,117 cells; Scale bar = 5 µm. h Representative genome browser tracks of Cut&Run assay with E2f4 antibodies in untreated and HU-treated (4 h) p19/Nras Trim33-WT cells, expressing HA-Recql. The upper two tracks show HA-Recql binding in the same cells. i Relationship between E2f4 and Recql recruitment at 2000 transcription start sites with maximal E2f4 enrichment after HU treatment in Trim33-WT cells. The regions were sorted by E2f4 tags and binned at 50 genes/bin followed by linear regression analysis. j PLA assay with antibodies to PCNA and pS5-RNAPII or total RNAPII in untreated or HU-treated (4 h) p19/Nras Trim33-WT and Trim33-KO cells. From left, n = 50,50,50,46,39,46,50,50 cells. Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. b , g , j Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunoprecipitation, Labeling, Sequencing, Transfection, Control, Two Tailed Test, Expressing, Binding Assay

Journal: Nature Communications

Article Title: Trim33 masks a non-transcriptional function of E2f4 in replication fork progression

doi: 10.1038/s41467-023-40847-0

Figure Lengend Snippet: a Immunofluorescence analysis with 53bp1 antibodies in p19/Nras (Ctrl) and p19/Nras/Myc (Myc) cells expressing shCtrl or shTrim33. At least 24 cells were quantified. Scale bar = 2 μm. b Immunofluorescence analysis with pH2AX antibodies in p19/Nras/Myc cells expressing shTrim33 or shCtrl and siRNAs against E2f4, Recql or control. At least 133 cells were quantified per group. Scale bar = 5 μm. c Neutral comet assays in p19/Nras/Myc cells expressing shRNAs against Trim33 or a control sequence and siRNAs against E2f4, Recql, or control. Scale bar = 10 μm. At least 39 cells were quantified per group. d Schematic of the HDTV-based model of liver tumorigenesis. Transposon vectors encoding Myc, Nras, GFP, and shCtrl or shTrim33 are injected along with the SB transposase in the tail vein of C57BL/J mice. e Representative images of whole livers under daylight and ultraviolet light showing tumor nodules. f Kaplan–Meier survival plots for two cohorts of mice injected with shTrim33 or shCtrl (7 animals/group). Significance was determined using the log-rank test. g Representative images of IHC analysis with antibodies to Trim33 and pH2AX on FFPE sections of tumor-bearing livers. h Quantification of pH2AX-positive cells per view field in liver tumors expressing shTrim33 or shCtrl. Significance was determined by a two-tailed, unpaired t -test. a , b , c Significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons. a , b , c , h Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.

Article Snippet: E2f4-GST fusion proteins were generated by cloning the PCR-amplified E2f4 cDNA fragments (1–413, 1–105, 90–200, 300–413) into the pGEX-4T3 vector using the HiFi assembly kit (Cell Signaling).

Techniques: Immunofluorescence, Expressing, Control, Sequencing, Injection, Two Tailed Test