Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: ETV2 binds with KDM4A (A and B) Rosa26-CreERT2;Kdm4a/4c f/f mESCs ±4′-OH-tamoxifen were differentiated and analyzed on day 4 for RT-qPCR (A, n = 3) and flow cytometry (B, B′, n = 3). (B) Representative data from three independent experiments. (B′) Quantification data of flow cytometry. Con, wild-type control; DKO, double knockout; FL1, empty channel of flow cytometry. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C) Overexpression of ETV2 increases genes downregulated in Kdm4a/4c DKO mESCs. Kdm4a/4c DKO mESCs overexpressing Etv2 were differentiated and subjected to RT-qPCR. n = 3. ∗∗∗ p < 0.001. (D) Enhanced H3K9me3 in endothelial and hematopoietic genes in the absence of ETV2. Etv2 −/− mESCs were differentiated, harvested at day 4 and cross-linked. Subsequently, the nuclear genomic DNAs were sonicated, and the fragmented genomic DNAs were immunoprecipitated with rabbit anti-mouse H3K9me3 antibody or rabbit IgG antibody. The immunoprecipitated DNA fragments were qPCR-amplified with primers corresponding to the promoter or enhancer regions of the indicated genes. DNA enrichment is shown as a percentage of input DNA. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05. (E–G) (E) Interaction of ETV2 and KDM4A. HEK/293T cells transfected with the indicated constructs were subjected to immunoprecipitation (IP) and western blot analysis. Anti-TUBULIN antibody was used for the internal loading control. (F) In vitro translated FLAG-ETV2 and HA-KDM4A proteins were subjected to immunoprecipitation with an anti-FLAG antibody and western blot analysis with the indicated antibodies. (G) The differentiated wild type mESCs were subjected to immunoprecipitation with an anti-KDM4A antibody, followed by a WB blot analysis with an anti-ETV2 antibody. (H) The cooperative function of ETV2-KDM4A interaction in inducing Flk1 promoter/enhancer ( p/e ) activity. Each construct expressing ETV2 or KDM4A (wt or H188A, a defective mutant of demethylation activity of KDM4A) was introduced into HEK/293T cells together with the Firefly luciferase reporter construct, pGL3-Flk1(p/e) . The Firefly luciferase activity was normalized with Renilla luciferase activity. Data are mean ± SEM of three independent experiments with triplicate assays/experiments. ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: PE-conjugated Rat anti-mouse FLK1/VEGFR2 antibody , Biolegend , Cat# 136403; RRID: AB_1967093.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control, Double Knockout, Over Expression, Sonication, Immunoprecipitation, Amplification, Transfection, Construct, Western Blot, In Vitro, Activity Assay, Expressing, Mutagenesis, Luciferase

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet: The ETV2-mediated generation of FLK1 + cells, hematopoietic and endothelial lineages is regulated by the demethylase activity of KDM4A (A and B) iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated for 4 days were subjected to flow cytometry (A, A′, n = 3) and gene expression analysis (B, n = 3). Dox (1 μg/mL) was treated at day 2. (A) Representative data from three independent experiments is shown. Numbers in the plots denote the percentages of FLK1 + cells. FL1: empty channel. (A′) The quantification data of the flow cytometry. (B) Expression of each gene was normalized against Gapdh , and the fold change of its expression level (+Dox/-Dox) was calculated. Data are mean ± SEM of three independent experiments with duplicate assays/experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, n.s.: not significant. (C and D) Flow cytometry analysis for FLK1 and hCD4/SCL (C, C′) and CDH5 (D, D′) in D6 EBs of indicated mESCs. Results are means ± SEM from three independent experiments. FL1: empty channel. ∗ p < 0.05, ∗∗ p < 0.01. (E) Hematopoietic colony assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with ± Dox at D3 and subjected to a hematopoietic replating assay at day 6. Colonies were counted 4 days later. Results are means ± SEM from three independent experiments. ∗∗ p < 0.01. (F) EB sprouting assay. iFLAG-ETV2 and iFLAG-ETV2-HA-KDM4A (H188A) mESCs differentiated under serum-free conditions were treated with Dox at D3 and subjected to the sprouting assay in a 3D-collagen matrix. The mean sprouting area was measured using ImageJ software 8 days later. 3 EBs/group, n = 3, ∗ p < 0.05.

Article Snippet: PE-conjugated Rat anti-mouse FLK1/VEGFR2 antibody , Biolegend , Cat# 136403; RRID: AB_1967093.

Techniques: Activity Assay, Flow Cytometry, Gene Expression, Expressing, Hematopoietic Colony Assay, Software

Journal: iScience

Article Title: ETV2/ER71 regulates hematovascular lineage generation and vascularization through an H3K9 demethylase, KDM4A

doi: 10.1016/j.isci.2024.111538

Figure Lengend Snippet:

Article Snippet: PE-conjugated Rat anti-mouse FLK1/VEGFR2 antibody , Biolegend , Cat# 136403; RRID: AB_1967093.

Techniques: Plasmid Preparation, Recombinant, Virus, Control, Transfection, Staining, Knock-Out, Protease Inhibitor, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Cell Culture, DNA Purification, DNA Methylation Assay, Reporter Assay, Software

Journal: STAR Protocols

Article Title: Protocol for the three-dimensional analysis of rodent skeletal muscle

doi: 10.1016/j.xpro.2024.103549

Figure Lengend Snippet: Perfusion fixation, tissue dissection and mounting EDL muscle (A) Following anesthetization, TMX-treated Pax7 tdTomato :Flk1 GFP mice were perfusion-fixed with a syringe pump. (B) Perfusion-fixed mice had their hindlimb skin excised and the EDL muscle detached from the hindlimb muscle. (C and D) Silicon isolator with cover glasses was used for tissue-mounting. (E and F) The tissue clearing process made the EDL muscle transparent (E), compared with the EDL muscle without tissue clearing (F).

Article Snippet: Flk1 GFP mice ( Kdr tm2.1Jrt /J ), 2 months old (male or female) , Jackson Laboratory , #017006.

Techniques: Dissection

Journal: STAR Protocols

Article Title: Protocol for the three-dimensional analysis of rodent skeletal muscle

doi: 10.1016/j.xpro.2024.103549

Figure Lengend Snippet:

Article Snippet: Flk1 GFP mice ( Kdr tm2.1Jrt /J ), 2 months old (male or female) , Jackson Laboratory , #017006.

Techniques: Recombinant, Saline, Mouse Assay, Software, Microscopy, Laser Capture Microdissection