Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . The CHD MPRA library included 6590 REF-ALT pairs. After pooled library synthesis of barcoded oligos, the oligos were PCR amplified and cloned into lentivirus genome backbone. A minimal promoter (miniP)-GFP cassette was then inserted into the cloned oligo library. b . Summary of activity of CHD MPRA library. Plot on bottom indicates the occurrence of the indicated annotation with a vertical line. Enrichment score represents enrichment of the indicated set of annotations at either end of the list of all regions, ranked by activity. Enrichment p-value was determined by 1-sided permutation test, with Bonferroni correction. Active enhancers had barcodes overrepresented in RNA compared to DNA (DESeq2 P adj < 0.05). c . Pearson correlation (PCC) between regions shared between the Mutagenesis MPRA and the CHD MPRA. The same genomic sequences had different barcodes in the two assays. d . Validation of the effect of variants on transcription factor binding. EMSA assay was used to test the binding of SRF or TBX20 to REF or ALT variant sequences. For the GLB1L3 CRE, ALT disrupted the SRF motif and reduced SRF binding in the EMSA assay. For the PIP4K2A CRE, ALT generated a TBX20 motif and increased TBX20 binding in the EMSA assay. Representative of three independent experiments. Two-tailed t-test. n = 3 per group. Graph shows mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Amplification, Clone Assay, Activity Assay, Mutagenesis, Genomic Sequencing, Biomarker Discovery, Binding Assay, Variant Assay, Generated, Two Tailed Test

Journal: Nature genetics

Article Title: Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease

doi: 10.1038/s41588-024-01669-y

Figure Lengend Snippet: a . BCOR downregulation in SMAD2 Het and KO iPSC-CMs. Gene expression was measured by RNA-seq. One-way ANOVA with Dunnett’s multiple comparison test versus WT. n = 3. b . Effect of ncDNVs on binding of transcription factors to CREs near CHD genes. 39 bp duplexes centered on ncDNVs neighboring 4 CHD genes were synthesized. Binding of purified, recombinant proteins to the REF or ALT sequence was measured by electrophoretic mobility shift assay (EMSA). SMAD2 and HIC2 bound CREs near BCOR and ACVRL1 more strongly for REF compared to ALT. In contrast, SRF and TBX20 bound CREs near ADAMTS6 and MYOCD more strongly for ALT compared to REF. Note lower free probe in MYOCD -ALT compared to REF. Results are representative of at least three independent experiments. Quantification of TBX20 EMSA: mean ± SD; n = 3; two-sided t-test. Graphs in a and b show mean ± SD.

Article Snippet: Recombinant human proteins used in this study included SMAD2 (Abcam, ab85329), SRF (OriGene, TP308596), TBX20 (OriGene, TP762422), HIC2 (OriGene, TP760963), SOX9 (OriGene, TP308944) and GATA4 (OriGene, TP310945).

Techniques: Gene Expression, RNA Sequencing, Comparison, Binding Assay, Synthesized, Purification, Recombinant, Sequencing, Electrophoretic Mobility Shift Assay

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then treated with actinomycin D for different periods of time. RT-qPCR assay analysis of vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels ( n = 5 independent experiments). b HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h, then transfected with various VEGFR2 promoter truncation constructs or a mutant (serum response factor [SRF] binding site) construct for 24 h. After this, luciferase activity was measured ( n = 5 independent experiments). c Chromatin immunoprecipitation assays using an anti-SRF antibody to amplify VEGFR2 promoter in HUVECs transfected with or without FUNDC1 siRNA ( n = 5 independent experiments). d HUVECs were pre-transfected with or without FUNDC1 siRNA for 24 h and then treated with VEGF for 30 min. Expression of SRF and phosphorylated SRF at Ser103 (pSRF) was determined by western blot assay ( n = 5 independent experiments). e Cytosolic Ca 2+ levels, as indicated by the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). HUVECs were transfected with Scr siRNA or FUNDC1 siRNA for 24 h and then exposed to VEGF (20 ng/mL) for the indicated times. f HUVECs were infected with adenovirus encoding control (Ad-Cont) or mitochondrial–endoplasmic reticulum (ER) linker (Ad-Linker) for 24 h and then treated with BAPTA-AM (10 µM) or control dimethylsulfoxide (vehicle) for 30 min. Levels of SRF and pSRF (Ser103) were detected by western blot assay ( n = 5 independent experiments). g HUVECs were pre-transfected with or without SRF siRNA for 24 h, then infected with adenovirus encoding control or mitochondria–ER linker for 24 h. VEGFR2 protein expression was detected by western blot assay ( n = 5 independent experiments). Statistical significance was assessed using two-tailed t -tests for two groups and one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Scr siRNA; Scr siRNA+VEGF; Ad-Cont+Vehicle or Ad-Cont+Scr siRNA. All values are mean ± S.D.

Article Snippet: SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas).

Techniques: Transfection, Quantitative RT-PCR, Construct, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Chromatin Immunoprecipitation, Expressing, Western Blot, Infection, Control, Two Tailed Test

Journal: Nature Communications

Article Title: FUNDC1-dependent mitochondria-associated endoplasmic reticulum membranes are involved in angiogenesis and neoangiogenesis

doi: 10.1038/s41467-021-22771-3

Figure Lengend Snippet: a Transduction efficiency of peptides 1, 2, and a control peptide in Human umbilical vein endothelial cells (HUVECs) ( n = 5 independent experiments). b HUVECs were treated with different peptides (20 μM) for 24 h, then subjected to immunoprecipitation with antibody against FUNDC1(HA) to quantify the interaction of FUNDC1 and IP3R1 ( n = 3 independent experiments). c HUVECs were treated with different peptides (20 μM) for 24 h, then cytosolic Ca 2+ was detected using the fluorescent probe Fluo-4, AM ( n = 5 independent experiments). d HUVECs were treated with different peptides. After 24 h, the cell lysates were collected and subjected to western blot assays to detect the expression of VEGFR2, IP3R1, SRF, and phosphorylated SRF at Ser103 (pSRF) ( n = 5 independent experiments). e Three-dimensional spheroids and representative images of spheroid-sprouting were analyzed ( n = 5 independent experiments). Scale bar, 100 µm. f Matrigel containing vascular endothelial growth factor (VEGF) was injected subcutaneously into 6-week-old wild-type mice, then the mice received an intravenous injection of the indicated peptides. After 10 days, matrigel plugs were removed for analysis of new vessel formation by histological and Hb assay ( n = 5 mice/group). Quantification of Hb extracted from matrigel plugs of different groups. g C57BL/6 mice with LLC tumors that were approximately 90 mm 3 in size were treated with intravenous injections of different peptides (10 mg/kg/3 days). After 28 days, the tumors were harvested and quantified. Representative images of tumors ( n = 5 mice/group). h Immunostaining of LLC tumor sections and quantification of relative CD31-positive area. ( n = 5 mice/group). Scale bar, 100 µm. Statistical significance was assessed using one-way ANOVA with post hoc multiple comparisons test for multiple groups. *p < 0.05 vs Ctrl peptide. All values are mean ± S.D.

Article Snippet: SiRNA for FUNDC1, IP3R1, VEGFR2, serum response factor (SRF), and controls were obtained from Santa Cruz Biotechnology (sc-36563, sc-91118, sc-42475, and sc-29318; Dallas, Texas).

Techniques: Transduction, Control, Immunoprecipitation, Western Blot, Expressing, Injection, Immunostaining

Journal: Nature communications

Article Title: Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

doi: 10.1038/ncomms10830

Figure Lengend Snippet: Figure 7 | Nuclear PTEN blocks PDGF-mediated repression of SM gene transcription. (a,b) SMCs were serum-restricted for 48 h followed by stimulation with vehicle control or 20 ng ml 1 PDGF-BB for 24 h (a) or 48 h (b). (a) SMCs were fixed, immunofluorescently stained for PTEN (green) and analysed for PTEN localization using confocal microscopy; nuclei were stained for DAPI (blue). (b) PTEN was immunoprecipitated (IP) from cytoplasmic (cyto) and nuclear (nuc) fractions of vehicle- or PDGF-stimulated SMCs. Co-immunoprecipitating SRF was detected by immunoblotting (IB). Representative western blot from three separate experiments. (c) SMCs were transfected with a construct expressing SRF–GFP, maintained in serum-restricted conditions or stimulated with 20 ng ml 1 PDGF-BB, fixed and analysed for GFP localization; nuclei were stained for DAPI (blue). Shown are representative images (two serum-restricted and four PDGF-stimulated cells are shown); arrows indicate cytoplasmic localized SRF–GFP; nuclei are outlined with white lines. (d) SMCs were transfected with HA-tagged wild-type PTEN (WT), nuclear localized PTEN (NLS) or nuclear excluded PTEN (NES). SMCs were maintained in serum-restricted conditions or stimulated with 20 ng ml 1 PDGF-BB, fixed, immunofluorescently stained for HA (red) and analysed for PTEN localization; nuclei were stained for DAPI (blue). Arrowheads, HA–PTEN-transfected SMCs. (e) SMCs were transfected with GFP–SRF (ctrl) or co-transfected with GFP–SRF and WT PTEN or nuclear localized PTEN (NLS) then maintained in serum-restricted conditions or stimulated with 20 ng ml 1

Article Snippet: Plasmid-encoding human SRF tagged with GFP (RG208596) was obtained from OriGene.

Techniques: Control, Staining, Confocal Microscopy, Immunoprecipitation, Western Blot, Transfection, Construct, Expressing

Journal: Cell metabolism

Article Title: Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta-cell proliferation

doi: 10.1016/j.cmet.2017.04.014

Figure Lengend Snippet: A. Heatmap showing Pearson correlation of gene expression profiles in all 387 beta-cells comparing proliferation genes with top pseudotemporally-regulated oxidative phosphorylation genes and TFs. Proliferation genes are depicted in red, oxidative phosphorylation genes in orange and TFs in black. B. Overview of RNA-seq analysis of lentiviral Srf overexpression in islets at P28. C. GSEA plots showing enrichment of proliferation genes regulated during pseudotime (left) and genes down-regulated during pseudotime (right) as an effect of Srf overexpression. RNA-seq data are from three independent transduction experiments. D. RPKM values of TFs Fos, Junb and Egr1 (top panel) and proliferation genes Mki67, Pcna and Ccne1 (bottom panel) in RNA-seq data from control and Srf-overexpressing islets. Data shown as mean ± SEM. E. Summary of metabolic regulators and effector TFs driving early neonatal beta-cell proliferation as revealed by reconstructing a pseudotemporal time course of beta-cell maturation, experimental validation, and prior literature. ** P < 0.01, *** P < 0.001. See also Figure S7 and Table S7.

Article Snippet: Lentivirus production and transduction GFP-tagged lentiviral plasmid (Origene PS100071) or GFP-tagged Srf lentiviral plasmid (Origene MR208120L2) was transfected with pCMV-R8.74 (Addgene 22036) and pMD2.G expression plasmid into HEK293T cells.

Techniques: Expressing, RNA Sequencing Assay, Over Expression, Transduction

Journal: Cell metabolism

Article Title: Pseudotemporal ordering of single cells reveals metabolic control of postnatal beta-cell proliferation

doi: 10.1016/j.cmet.2017.04.014

Figure Lengend Snippet: Key Resources Tables

Article Snippet: Lentivirus production and transduction GFP-tagged lentiviral plasmid (Origene PS100071) or GFP-tagged Srf lentiviral plasmid (Origene MR208120L2) was transfected with pCMV-R8.74 (Addgene 22036) and pMD2.G expression plasmid into HEK293T cells.

Techniques: Recombinant, Staining, Sequencing, Imaging, In Situ, Enzyme-linked Immunosorbent Assay, Sample Prep, Real-time Polymerase Chain Reaction, Software