Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A FACS analysis of the percentage of GFP + (Cas9 + ) cells within the mCherry + (sgRNA + ) and mCherry - (sgRNA - ) HSPCs on day two (top) and six (bottom) post transduction. B Heatmap of survival/proliferation scores in HSPCs treated with the indicated sgRNAs ( n = 2, biological replicates). The color indicates decreased (orange) and increased (blue) survival/proliferation scores. C Western blot of TFDP1 and E2F4 three days post targeting with sgRNAs against Tfdp1 and E2f4 . SgRosa26-1 was used as a negative control. Actin was used as a loading control. D Co-immunoprecipitation (Co-IP) using TFDP1 (top) and E2F4 as precipitating antibody (bottom) ( n = 2, biological replicates).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Transduction, Western Blot, Negative Control, Control, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A Scheme of assessment of HSPC proliferation and apoptosis. Cas9-HSPCs were labeled with Celltrace, cultured for one day and transduced with lentiviral particles expressing sgRNAs targeting Tfdp1 , E2f4 , or the Rosa26 . B Representative FACS analysis of the percentages of Annexin V + DAPI - (early) and Annexin V + DAPI + (late) apoptotic cells within mCherry - (sgRNA - , upper panel) or mCherry + (sgRNA + , lower panel) HSPCs treated with the indicated sgRNAs. C Representative FACS analysis of active Caspase 3 + apoptotic HSPCs treated with the indicated sgRNAs three days post puromycin selection (top) and summary of the data (bottom) based on HSPCs from three mice ( n = 3). D Representative FACS analysis of the proliferation rates of mCherry - (sgRNA-) and mCherry + (sgRNA + ) HSPCs infected with the indicated sgRNAs two and four days post cell-trace labeling. The number of cell divisions is indicated. E Percentage of cell division in mCherry + (upper) and mCherry - (below) HSPC subpopulations treated with the indicated sgRNAs on day two and day four post cell-trace labeling ( n = 3 independent experiments).

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Labeling, Cell Culture, Transduction, Expressing, Selection, Infection

Journal: Leukemia

Article Title: In vivo CRISPR/Cas9-mediated screen reveals a critical function of TFDP1 and E2F4 transcription factors in hematopoiesis

doi: 10.1038/s41375-024-02357-w

Figure Lengend Snippet: A Venn diagrams depicting the overlap between the differentially expressed genes in Tfdp1 -KO HSPCs (downregulated genes in blue and upregulated genes in red) and human TFDP1- (left; GSE80661; GSE105217; GSE127368), human E2F4- (middle; GSE31477; GSE170651), and mouse E2F4- (right; GSE48666) bound target genes. B Intersection between human TFDP1- and E2F4-bound genes downregulated in Tfdp1 -KO HSPCs. C Density plots (upper panel) and heatmaps (lower panel) depicting the average tag densities around TSSs (−2/+2 kb) of up- and downregulated genes in Tfdp1 KO HSPCs. Data are derived from the ChIP-seq of RNA polymerase II S5P (RnapolII S5P; GSE34518), H3K4Me3 (GSE75426), and E2F4 (GSE48666) (together with a negative control) in mouse ES cells. Right panel: ATAC-seq signals (GSE100738) from mouse short-term (ST) HSCs in the same genomic regions. D Example of RnapolII S5P, H3K4Me3, E2F4 tracks in mouse ES cells and ATAC-seq in ST-HSCs at the mouse Cdk1 locus. E Example of E2F4 and TFDP1 ChIP-seq signals at the CDK1 locus in various human cell types. Mouse and human E2F4 and TFDP1 DNA binding sites derived from the Unibind database are shown and the core nucleotides involved in DNA binding are highlighted.

Article Snippet: TFDP1, E2F4, E2F1, and beta-Actin proteins were detected using primary antibodies: mouse anti-TFDP1 (Thermo Scientific, Cat# MA5-11268), mouse anti-E2F4 (Proteintech, Cat# 67812-1-Ig), rabbit anti-E2F4 (Sigma, Cat# AV31175), mouse anti-E2F1 (Proteintech, Cat# 66515-1-Ig) and mouse anti-β-Actin (Sigma-Aldrich Cat# A2228).

Techniques: Derivative Assay, ChIP-sequencing, Negative Control, Binding Assay

Journal: Nucleic acids research

Article Title: A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

doi: 10.1093/nar/gkae370

Figure Lengend Snippet: Figure 2. A-MYB regulates Cyclin B2 by indirectly binding to its CHR promoter element through MuvB. ( A ) siRNA knockdown was performed in HCT116 ( n = 5) and U2OS ( n = 4) cells. Tw enty -f our hours af ter knoc kdo wn, cells w ere transfected with luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates. Differences between B-MYB KD and A-MYB + B-MYB dKD are statistically not significant. ( B ) To rescue HCT116 cells from siRNA knockdown of A-MYB and B-MYB , cells were transfected with the mouse A-Myb-overexpressing plasmid mA-Myb-GFP or the empty vector pEGFP as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter in parallel to the siRNA transfections. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 3). ( C ) For luciferase assa y s after mouse A-Myb o v ere xpression, HCT116, RPE-1, Hep3B or HeLa cells were transfected with the mAmyb-GFP overexpression vector as well as luciferase reporter constructs carrying the wild-type (wt) or CHR-mutated (CHRmut) Cyclin B2 promoter. Forty-eight hours after knockdown, luciferase activity was quantified from cell lysates ( n = 12). ( D ) Binding of A-MYB to LIN37 was tested in CoIP assays with HCT116 native protein extracts. As controls, E2F4 representing the DREAM complex and B-MYB as MMB component were precipitated (representative experiment of n = 4). ( E ) Binding of A-MYB to the MuvB core components LIN9, LIN37 and LIN54 was tested in CoIP assays with RPE-1 native protein extracts. As controls, LIN9 and LIN37 representing the MuvB core complex and B-MYB as an MMB component were precipitated (representative experiment of n = 3). ( F ) Binding of the MuvB core to A-MYB was tested in CoIP assa y s with RPE-1 native protein extracts. As negative control, p130 representing the DREAM complex was precipitated (representative experiment of n = 3). ( G ) T98G cells were synchronized in the cell cycle by density arrest and released for 15 h or 24 h into the cell cycle to obtain cell populations enriched in G 0 , S and G 2 / M phase, respectively. From each time point, native protein extracts were isolated and subjected to a LIN37 CoIP. Binding of DREAM and MMB components B-MYB, E2F4, and LIN37 as well as A-MYB was analyzed by Western blot (representative from n = 3). ( H ) Binding of A-MYB to wild-type (wt) and CHR mutant (CHRmut) Cyclin B2 promoter probes in DNA-affinity purification was analyzed by Western blot (representative of n = 3). Mean ± SD are given, and significances were calculated by two-way ANO V A (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: For protein detection, these antiodies were used: β-actin (A5441, Sigma-Aldrich), A-MYB HPA008791, Sigma-Aldrich), B-MYB (A 301–655A, Bethyl aboratories / Fortis), CDC25C (H-6, Santa Cruz Biotechology), Cyclin B2 (A-2, Santa Cruz Biotechnology), E2F4 E3G2G rabbit mAb, 40291, Cell Signaling Technology), hisone 3 (D2B12 XP® rabbit mAb, 4620S, Cell Signaling Techology), LIN9 (A300-BL2981, Bethyl Laboratories / Fortis), IN37 (T3, custom-made at Pineda Antikörper-Service, erlin, Germany) (Müller et al., 2016), LIN54 (A303-799A, ethyl Laboratories / Fortis), Survivin (71G4B7, Cell Signaling echnology).

Techniques: Binding Assay, Knockdown, Transfection, Luciferase, Construct, Activity Assay, Plasmid Preparation, Over Expression, Negative Control, Isolation, Western Blot, Mutagenesis, Affinity Purification